Pre-harvest sprouting

ABSTRACT

The invention relates to materials and methods which may be used in the detection and manipulation of Pre-Harvest Sprouting (PHS) and other dormancy-related phenotypes in plants. Specifically disclosed are oat and wheat VP1 homologues (afVP1 and taVP1 respectively) plus also variants, particularly alleles of these. The sequence and mapping data provided can be used in plant breeding and/or in molecular-biology based methods to improve e.g. wheat varieties. Also disclosed are primers which are specific for orthologues, alleles or wheat-genomes plus methods of using these. Vectors, cells and transgenic plants are also provided, as are related products and methods of use.

TECHNICAL FIELD

The present invention relates to materials and methods which may be usedin the detection and manipulation of Pre-Harvest Sprouting (PHS) andother dormancy-related phenotypes in plants. The present invention alsorelates to materials and methods for use in plant breeding—in particularto molecular-biology based methods for generating, identifying,characterising or manipulating genetic variation which affects the PHSand other dormancy related traits.

PRIOR ART Pre-Harvest Sprouting (PHS)

Pre-Harvest Sprouting (PHS) of non-dormant grains is a major limitingfactor in achieving consistent bread making quality of UK wheat. Averageannual losses due to PHS in the UK wheat crop have been estimated atsome £17 million, but the problem is erratic and is much more severe incool, damp seasons. Variation in the degree of sprouting damage fromyear to year makes this problem difficult to select against inconventional breeding programmes.

VP1 in Maize and Other Species

Previous work in other plant species has shown that the VIVIPAROUS 1gene is a major regulator of embryo maturation in maize. Thus McCarty etal (1989) in The Plant Cell 1, 523-532 disclosed that vp1 mutants inmaize were abscisic acid (ABA) insensitive, and demonstrated its role incontrolling the developmental responses associated with the maturationphase of seed formation. VP1 mutants were shown to germinateprecociously. Similarly McCarty et al (1991) in Cell 55, 895-905disclosed that VP1 encoded 73 kDa transcription factor.

Giraudat et al (1992) in The Plant Cell 4, 1251-1261 showed thatArabidopsis ABI3 mutants had altered seed development & germination. Thepredicted gene product was similar to VP1 protein. These results andothers show that ABI 3/VP1 function as developmental regulators duringthe maturation stage of embryogenesis by regulating transcription ofsets of genes that determine the embryonic phenotype in preparation fordesiccation of the seed prior to shedding.

A VP1 homolog in rice has also been isolated (Hattori et al (1994) PlantMolecular Biology 24, 805-810). Similarly in Abstract, Poster No P184“Poaceae sequence analysis: cloning of a VP-1 homolog from genomicbarley DNA via PCR”, at the Plant and Animal Genome V Conference in SanDiego, USA, January 1997, Wilson & Sorrells disclosed the use ofconserved primers to pick out vp1 homologs in Barley.

Wilson speculated that a wheat VP1 homolog may be obtainable bycomparison with maize, rice and barley sequences, and (on the basis ofcomparison with these species) that the R locus may contain a wheat VP1homolog.

Interestingly, earlier work by Cadle et al (1994) in Genome 37, 129-132had already shown that the maize vp1 from McCarty didn't hybridisestrongly to wheat DNA and could not therefore be used as a probe to mapthe wheat gene, although various ABA-inducible genes were mappedsuccessfully.

Dormancy

Other recent studies of the genetics of the transition fromembryogenesis to germination in maize and Arabidopsis show that mutationof GA (gibberellic acid) and ABA synthesis and sensitivity can alterdormancy levels (Koornneef and Karssen, 1994). For example, whereas theArabidopsis mutation ga 1 causes a loss of germination due to GAdeficiency, aba/abi mutations (that affect ABA synthesis and perceptionrespectively) cause a loss of dormancy (and in strong alleles, loss ofviability) because embryos fail to develop desiccation tolerance duringmaturation (for example alleles of abi 3, Ooms et al., 1993).

It has been suggested that PHS in wheat is the result of the lack ofinduction of dormancy during embryo development (Gale and Lenton 1987).As is known to those skilled in the art, dormancy is one of two possibledevelopmental states which mature seeds may show following desiccationand shedding (the other being germination). Embryo dormancy developsduring late embryogenesis, and results in a lack of germination.Following imbibition of the mature shed seed it results in an inactivephase of plant growth during which development is deferred, although theembryo still maintains a high metabolic activity. Dormancy of matureimbibed seeds occurs even under environmental conditions that wouldfavour germination, indicating that the process is not simply a lack ofcorrect conditions. During dormancy, cells within the mature embryo aremaintained in an arrested state, and nuclear DNA values obtained from A.fatua embryos indicate that the cell-cycle is held in G1 and DNAreplication does not occur during imbibed dormancy (Elder and Osborne1993). Dormancy is probably an evolutionary strategy that allowssurvival of seeds through adverse conditions, and dispersal of seedgermination through time. Dormancy is therefore a very important phaseof plant development required both for the inhibition of germinationprior to completion of embryogenesis, and for the pre-germinativesurvival of mature seeds. It is also an important agronomic trait, withthe market value of wheat being determined, inter alia, by its HagbergFalling Number, which measures the degree to which somegermination-related processes have progressed (discussed in relation toplant breeding hereinafter).

Seeds of the persistent weed A. fatua can show very high levels ofembryo dormancy (Simpson 1978).

Embryos with primary dormancy go through a time and environmentsensitive process of after-ripening in the dry seed, that is manifestedby loss of dormancy in the imbibed seed (Mayer and Poljakoff-Mayber1989). Dormancy can subsequently be reimposed on after-ripened dryembryos under specific environmental conditions (‘induced’ or‘secondary’ dormancy). These features indicate that signals perceived bythe dry seed influence developmental choices following imbibition,resulting in either dormancy or germination (Hilhorst and Karssen 1992).Recent work on the water status of embryos of A. fatua has demonstratedthat individual enzymatic and non-enzymatic reactions, rather thanmetabolic processes control this dormancy/non-dormancy switch in the dryseed (Foley 1994). Others have proposed that the process may involvekinase-phosphatase interactions (Trewavas 1987).

Many studies have analysed the genetic control of embryo dormancy(Hilhorst and Karssen 1992). Results obtained from experiments withinbred lines of A. fatua have suggested that in this species dormancymay be controlled by three loci, two that promote dormancy (L1 and L2),and one that promotes after-ripening (E) (Jana et al., 1979,

Jana et al. 1988). These dormancy genes have not been cloned orcharacterised, their existence was inferred from statistical analysis ofsegregation for dormancy phenotypes among the progeny from a crossbetween two different strains.

There is currently a requirement for materials and methods which haveutility in the identification or molecular tagging of the genesresponsible for PHS in wheat, or which could be used in the manipulationof the PHS trait in wheat or other plants.

Thus it can be seen that the provision of such materials or methodswould provide a contribution to the art.

DISCLOSURE OF THE INVENTION

The present inventors have for the first time identified a gene from theoat Avena fatua which encodes a protein which has a high degree ofsimilarity to known VP1/ABI 3 related transcription factors. Theexpression product of this gene has been termed afVP1.

By studying imbibed mature seeds, the expression of afVP1 has beencorrelated with the dormant phenotype (primary dormancy, secondarydormancy and after-ripening) in oat. In particular, the presentinventors have demonstrated that wild oat has the potential forextremely high levels of dormancy in the mature dry seed, and thatexpression of afVP 1 is absolutely correlated to the dormant phenotypein imbibed mature seeds. This is the first demonstration of expressionof a VP 1-homologue in a developmental situation other thanembryogenesis. It indicates that afVP1 activity keeps mature seedsdormant, and inhibits germination—it can thus be used to maintain orimpose sufficient intensity and duration of dormancy to avoid PHS beforeharvest.

In addition to establishing an important role for afVP1 in the controlof after-ripening and both primary and secondary dormancy. The presentinventors have also employed afVP1 to identify the hitherto unobtainedwheat VP1 homolog (hereinafter taVP1) and map its genomic position. Aswill be discussed in more detail below, the information made availableby the present invention has important and industrially applicableimplications for the detection and manipulation of PHS and otherdormancy related traits in plants, and especially PHS in wheat. Inparticular work done by the inventors indicates that the ability to keepmature seeds dormant, and inhibit germination, has been lost by thewheat VP 1 due to breeding since domestication which has favoured theevolution of a crippled wheat VP 1 that cannot impose high levels ofdormancy (resistance to PHS) on the mature seed.

Introduction of the wild oat afVP 1 into wheat can therefore be used toinduce higher levels of dormancy (and thus resistance to PHS) in wheatas afVP 1 compensates for the crippled function of wheat VP 1.

Thus in a first aspect of the present invention there is provided anucleic acid molecule, encoding afVP1, and having the sequence set outin Seq ID No 1 (shown in FIG. 4 (a)). A further afVP1 sequence,differing slightly from Seq ID No 1, has been deposited in the EMBLdatabase under accession number AFJ001140 after the priority date of thepresent application.

The existence of an oat homologue to maize VP1 was reported briefly in aposter by M. J. Holdsworth “Dormancy-related expression of the wild oat(Avena fatua) homolog of the maize gene Viviparous 1 (Vp1)”. Abstract,Poster No.49, Seventh International Symposium on Pre-Harvest Sproutingin Cereals 1995, Abashiri, Japan, July 1995.

Some comments about its properties were made—however the precise meansof cloning the homolog, its sequence, and specific applications for it,were not disclosed.

Additionally Jones et al in April 1997 disclosed that afVP1 wascorrelated with primary and secondary dormancy (see Jones et al (1997) JExp Bot 48 (Suppl) 45). Once again no details about how afVP1 could beobtained, or particular applications for it, were disclosed.

The nucleic acid molecules and their encoded polypeptide products (seebelow) according to the present invention may be provided isolatedand/or purified from their natural environment, in substantially pure orhomogeneous form, or free or substantially free of nucleic acid or genesof the species of interest or origin other than the sequence encoding apolypeptide with the required function. Nucleic acid according to thepresent invention may include cDNA, RNA, genomic DNA and may be whollyor partially synthetic.

The term “isolate” encompasses all these possibilities. Where a DNAsequence is specified, e.g. with reference to a figure, unless contextrequires otherwise the RNA equivalent, with U substituted for T where itoccurs, is encompassed.

By virtue of its demonstrated properties, the nucleic acid of the firstaspect may has utility, inter alia, in producing transformed crop plantshaving desirable primary or secondary dormancy, or after-ripening,properties, and in particular may be resistant to PHS.

In a further (second) aspect of the invention there is disclosedvariants of the sequence provided, which may for instance be mutants orother derivatives, or naturally occurring alleles (or other homologues,including orthologues) of the sequence.

In the case of mutants and derivatives, the variant encodes a productwhich is homologous to the sequence of Seq ID No 1, and preferably whichretains a functional characteristic that the product encoded by thevariant has the afVP1 activity.

By ‘afVP1 activity’ is meant the ability to act as a transcriptionfactor which is capable of activating some or preferably all of thegenes which are activated by afVP1 (e.g. Em, C1) and repressing geneswhich are repressed by afVP1 (e.g. alpha-amylase—see Hoeker et al 1995for maize VP1 activity in this regard). This can be assayed eitherdirectly using e.g. a reporter gene system linked to any of these genesor their promoters. Alternatively it may be assayed by preparingtransformed plants and assaying its phenotypic effects in vivo (i.e.alteration of dormancy as described above).

Methodology for such transformation is described in more detailhereinafter.

Methods for producing such mutants or derivatives based on the sequenceprovided, and for identifying alleles (or other homologs) and thenassessing homology are discussed below, and form one part of this aspectof the invention.

In all cases the nucleic acid molecule which is the mutant or otherderivative is ultimately generated either directly or indirectly (e.g.via one or more amplification or replication steps) from oat afVP1(including alleles thereof), preferably from a nucleic acid moleculecomprising all or part of sequence ID No 1.

Changes to a sequence, to produce a mutant or derivative, may be by oneor more of addition, insertion, deletion or substitution of one or morenucleotides in the nucleic acid, leading to the addition, insertion,deletion or substitution of one or more amino acids in the encodedpolypeptide. Specifically included are parts or fragments (howeverproduced) corresponding to portions of the sequences provided, and whichencode polypeptides having afVP1 activity.

Changes may be desirable for a number of reasons, including introducingor removing the following features: restriction endonuclease sequences;other sites which are required for post translation modification;cleavage sites in the encoded polypeptide; motifs in the encodedpolypeptide for post translational modification. All of these may assistin efficiently cloning and expressing an active polypeptide inrecombinant form (as described below).

Other desirable mutation may be random or site directed mutagenesis inorder to alter the activity (e.g. specificity) or stability of theencoded polypeptide. Particular regions of interest may be those whichcorrespond to the regions of VP 1 which have been shown to function aseither a transcriptional activation domain (amino acids 28-121, McCartyet al. 1991), or as a repressor domain (amino acids 238-375, Hoecker etal. 1995). Sections of these regions are highly conserved amongst allthe VP 1 homologues (see FIG. 4, and discussion in the Examples)indicating that these sections may provide function to these regions.Comparison of the BR2 region, (shown previously to interact with otherclasses of transcription factors [Hill et al. 1996]), shows a highdegree of similarity between afVP 1 and other homologues, indicating aconservation of function for this part of the protein. These comparisons(in terms of predicted amino acid structure) are shown in FIG. 4.

Those regions which differ from the corresponding parts of other VP1 smay also be of interest in that they may be responsible for the highdormancy demonstrated for oat.

Specifically embraced are derivatives which are truncated, or which havefunctional regions replaced with corresponding regions from othersources.

As is well-understood, homology at the amino acid level is generally interms of amino acid similarity or identity.

Similarity allows for conservative variation, i.e. substitution of onehydrophobic residue such as isoleucine, valine, leucine or methioninefor another, or the substitution of one polar residue for another, suchas arginine for lysine, glutamic for aspartic acid, or glutamine forasparagine. As is well known to those skilled in the art, altering theprimary structure of a polypeptide (in this case the afVP1 protein) by aconservative substitution may not significantly alter the activity ofthat peptide because the side-chain of the amino acid which is insertedinto the sequence may be able to form similar bonds and contacts as theside chain of the amino acid which has been substituted out. This is soeven when the substitution is in a region which is critical indetermining the peptides conformation.

Also included are homologs generated from afVP1 having non-conservativesubstitutions. As is well known to those skilled in the art,substitutions to regions of a peptide which are not critical indetermining its conformation may not greatly affect its activity becausethey do not greatly alter the peptide's three dimensional structure. Inregions which are critical in determining the peptides conformation oractivity such changes may confer advantageous properties on thepolypeptide. Indeed, changes such as those described above may conferslightly advantageous properties on the peptide (e.g. altered stability,or DNA binding efficiency).

Similarity or homology (or identity, the terms are used synonymously)may be as defined and determined by the TBLASTN program, of Altschul etal. (1990) J. Mol. Biol. 215: 403-10, which is in standard use in theart, or, and this may be preferred, the standard program BestFit, whichis part of the Wisconsin Package, Version 8, September 1994, (GeneticsComputer Group, 575 Science Drive, Madison, Wis. USA, Wisconsin 53711)BestFit makes an optimal alignment of the best segment of similaritybetween two sequences. Optimal alignments are found by inserting gaps tomaximize the number of matches using the local homology algorithm ofSmith and Waterman.

Thus a mutant, derivative or allele (or other homolog) of the presentinvention shares homology with afVP1. Homology may be at the nucleotidesequence and/or amino acid sequence level. Preferably, the nucleic acidand/or encoded amino acid sequence shares homology with the codingsequence or the sequence encoded by the nucleotide sequence of Seq ID No1, preferably at least about 50%, or 60%, or 70%, or 80% homology, mostpreferably at least about 90%, 95%, 96%, 97%, 98% or 99% homology.

Homology may be over the full-length of the relevant sequence shownherein, or may more preferably be over a contiguous sequence of about orgreater than about 20, 25, 30, 33, 40, 50, 67, 133, 167, 200, 233, 267,300, 333, 400, 450, 500, 550, 600 or more amino acids or codons,compared with the relevant amino acid sequence or nucleotide sequence asthe case may be.

Thus a variant amino acid sequence in accordance with the presentinvention may include within the sequence shown in FIG. 4, a singleamino acid change with respect to the sequence shown in FIG. 4, or 2, 3,4, 5, 6, 7, 8, or 9 changes, about 10, 15, 20, 30, 40 or 50 changes, orgreater than about 50, 60, 70, 80 or 90 changes. In addition to one ormore changes within the afVP1 amino acid sequence shown in FIG. 4, avariant amino acid sequence may include additional amino acids at theC-terminus and/or N-terminus. Naturally, changes to the nucleic acidwhich make no difference to the encoded amino acid sequence (i.e.‘degeneratively equivalent’) are included.

A further part of the present invention provides a method of identifyingand cloning further afVP1 homologues or alleles from plant species whichmethod employs a nucleotide sequence as described above.

As mentioned earlier, the present inventors have already used thismethod to identify a hitherto unidentified wheat VP1 homolog (termedtaVP1)—this notwithstanding the failure of earlier workers (e.g. Cadleet al (1994)) who used maize VP1 to probe wheat. Nucleic acid moleculesidentified using this method form one part of the second aspect of theinvention e.g. taVP1, as do mutants and derivatives of those genes.

The originally derived nucleotide sequences of various clones of taVP1are shown in FIGS. 6 and 8.

Later sequences for the fully sequenced clone (designated taVP1—referredto as Seq ID No 2) and clones 2, 3, 4, 5, 6, and 9 are shown in FIGS.10(a)-(g).

It is apparent the sequences shown that none of them encodes a fulllength protein i.e. they appear to be crippled to various degrees. Thishas important implications for improving dormancy in wheat, as regardsmolecular biology-based methods for transforming or breeding improvedplants.

It may be noted that McKibbin et al, in a poster at SEB meeting in April1997 reported using the wild oat homolog of VP1 in N-blots of wheat mRNAfrom various cultivars to demonstrate that expression levels of certainmRNAs were reduced in non-dormant phenotypes (see McKibbin et al (1997)J Exp Bot 48: (Suppl) 47). However no data regarding the sequence of theputative wheat (or oat) homolog(s) was presented.

When identifying homologs using the strategies below, if need be clonesor fragments identified in the search can be extended to produce fulllength molecules. For instance if it is suspected that they areincomplete, the original DNA source (e.g. a clone library, poly(A)RNAextracted from embryos) can be revisited to isolate missing portionse.g. using sequences, probes or primers based on that portion which hasalready been obtained to identify other clones containing overlappingsequence.

In one embodiment, nucleotide sequence information provided herein maybe used in a data-base (e.g. of ESTs) search to find homologoussequences, expression products of which can be tested for ability asdescribed below.

In a further embodiment, a homolog or allele in accordance with thepresent invention is also obtainable by means of a method whichincludes:

(a) providing a preparation of nucleic acid, e.g. a genomic or cDNAlibrary),

(b) providing a nucleic acid molecule having a nucleotide sequence shownin or complementary to a nucleotide sequence shown in Seq ID No 1preferably from within the coding sequence (i.e. encoding for the afVP1amino acid sequence shown in FIG. 4), most preferably the probe used isdistinctive or characteristic of afVP1 rather than other, known, VP1analogs,

(c) contacting nucleic acid in said preparation with said nucleic acidmolecule under conditions for hybridisation of said nucleic acidmolecule to any said gene or homologue in said preparation, andidentifying said gene or homologue if present by its hybridisation withsaid nucleic acid molecule.

“Distinctive” or “characteristic” regions of afVP1 can be determinedwith reference to comparisons with know VP 1 homologues, for instancethose shown in FIG. 4. Such regions and specific oligonucleotides arefound away from the conserved regions which are described in more detailin Example 2 below, and will allow those skilled on the art to designprobes and primers which will not hybridise with prior art sequences.

Probing may employ the standard Southern blotting technique. Forinstance DNA may be extracted from cells and digested with differentrestriction enzymes. Restriction fragments may then be separated byelectrophoresis on an agarose gel, before denaturation and transfer to anitrocellulose filter. Labelled probe may be hybridised to the DNAfragments on the filter and binding determined. DNA for probing may beprepared from RNA preparations from cells.

Test nucleic acid may be provided from a cell as genomic DNA, cDNA orRNA, or a mixture of any of these, preferably as a library in a suitablevector. If genomic DNA is used the probe may be used to identifyuntranscribed regions of the gene (e.g. promoters etc.), such as isdescribed hereinafter.

Preliminary experiments may be performed by hybridising under lowstringency conditions. For probing, preferred conditions are those whichare stringent enough for there to be a simple pattern with a smallnumber of hybridisations identified as positive which can beinvestigated further.

It is well known in the art to increase stringency of hybridisationgradually until only a few positive clones remain. Suitable conditionswould be achieved when a large number of hybridising fragments wereobtained while the background hybridisation was low. Using theseconditions nucleic acid libraries, e.g. cDNA libraries representative ofexpressed sequences, may be searched. Those skilled in the art are wellable to employ suitable conditions of the desired stringency forselective hybridisation, taking into account factors such asoligonucleotide length and base composition, temperature and so on.

For instance, screening may initially be carried out under conditions,which comprise a temperature of about 37° C. or less, a formamideconcentration of less than about 50%, and a moderate to low salt (e.g.Standard Saline Citrate (‘SSC’)=0.15 M sodium chloride; 0.15 M sodiumcitrate; pH 7) concentration.

Alternatively, a temperature of about 50° C. or less and a high salt(e.g. ‘SSPE’=0.180 mM sodium chloride; 9 mM disodium hydrogen phosphate;9 mM sodium dihydrogen phosphate; 1 mM sodium EDTA; pH 7.4). Preferablythe screening is carried out at about 37° C., a formamide concentrationof about 20%, and a salt concentration of about 5×SSC, or a temperatureof about 50° C. and a salt concentration of about 2×SSPE. Theseconditions will allow the identification of sequences which have asubstantial degree of homology (similarity, identity) with the probesequence, without requiring the perfect homology for the identificationof a stable hybrid e.g. 70% homology.

Suitable conditions include, e.g. for detection of sequences that areabout 80-90% identical, hybridization overnight at 42° C. in 0.25MNa₂HPO₄, pH 7.2, 6.5% SDS, 10% dextran sulfate and a final wash at 55°C. in 0.1×SSC, 0.1% SDS. For detection of sequences that are greaterthan about 90% identical, suitable conditions include hybridizationovernight at 65° C. in 0.25M Na₂HPO₄, pH 7.2, 6.5% SDS, 10% dextransulfate and a final wash at 60° C. in 0.1×SSC, 0.1% SDS.

Binding of a probe to target nucleic acid (e.g. DNA) may be measuredusing any of a variety of techniques at the disposal of those skilled inthe art. For instance, probes may be radioactively, fluorescently orenzymatically labelled. Other methods not employing labelling of probeinclude amplification using PCR (see below), RN′ase cleavage and allelespecific oligonucleotide probing. The identification of successfulhybridisation is followed by isolation of the nucleic acid which hashybridised, which may involve one or more steps of PCR or amplificationof a vector in a suitable host.

In a further embodiment, hybridisation of nucleic acid molecule to anallele or homologue may be determined or identified indirectly, e.g.using a nucleic acid amplification reaction, particularly the polymerasechain reaction (PCR). PCR requires the use of two primers tospecifically amplify target nucleic acid, so preferably two nucleic acidmolecules with sequences characteristic of afVP1 are employed. However,if RACE is used (see below) only one such primer may be needed.

PCR techniques for the amplification of nucleic acid are described inU.S. Pat. No. 4,683,195 and Saiki et al.

Science 239: 487-491 (1988). References for the general use of PCRtechniques include Mullis et al, Cold Spring Harbor Symp. Quant. Biol.,51:263, (1987), Ehrlich (ed), PCR technology, Stockton Press, N.Y.,1989, Ehrlich et al, Science, 252:1643-1650, (1991), “PCR protocols; AGuide to Methods and Applications”, Eds. Innis et al, Academic 795Press, New York, (1990).

Prior to any PCR that is to be performed, the complexity of a nucleicacid sample may be reduced where appropriate by creating a cDNA libraryfor example using RT-PCR or by using the phenol emulsion reassociationtechnique (Clarke et al. (1992) NAR 20, 1289-1292) on a genomic library.

Thus a method involving use of PCR in obtaining nucleic acid accordingto the present invention may include:

(a) providing a preparation of plant nucleic acid,

(b) providing a pair of nucleic acid molecule primers useful in (i.e.suitable for) PCR, at least one said primers having a sequence shown inor complementary to a sequence shown in Seq ID No 1 as described inabove,

(c) contacting nucleic acid in said preparation with said primers underconditions for performance of PCR,

(d) performing PCR and determining the presence or absence of anamplified PCR product. The presence of an amplified PCR product mayindicate identification of a gene of interest or fragment thereof.

Thus the methods of the invention may include hybridisation of one ormore (e.g. two) probes or primers to target nucleic acid. Where thenucleic acid is double-stranded DNA, hybridisation will generally bepreceded by denaturation to produce single-stranded DNA. Thehybridisation may be as part of a PCR procedure, or as part of a probingprocedure not involving PCR. An example procedure would be a combinationof PCR and low stringency hybridisation. A screening procedure, chosenfrom the many available to those skilled in the art, is used to identifysuccessful hybridisation events and isolated hybridised nucleic acid.

An oligonucleotide for use in probing or PCR may be about 30 or fewernucleotides in length (e.g. 18, 21 or 24). Generally specific (i.e.“distinctive” or “characteristic”) primers are upwards of 14 nucleotidesin length. For optimum specificity and cost effectiveness, primers of16-24 nucleotides in length may be preferred. Those skilled in the artare well versed in the design of primers for use processes such as PCR.If required, probing can be done with entire restriction fragments ofthe gene disclosed herein which may be 100's or even 1000's ofnucleotides in length.

Some preferred oligonucleotides have a corresponding to bases 1 to 892or 600 to 892 of Seq ID No 1. Primers may correspond to 1398 to 1417 or2272 to 2290 of Seq ID No 1 (which is 1410-1429 and 2285-2302 on theafVP1 sequence deposisted on the EMBL database under accession numberAFJ001140).

In a further (third) aspect of the present invention, the nucleic aciddescribed above is in the form of a recombinant and preferablyreplicable vector.

“Vector” is defined to include, inter alia, any plasmid, cosmid, phageor Agrobacterium binary vector in double or single stranded linear orcircular form which may or may not be self transmissible or mobilizable,and which can transform prokaryotic or eukaryotic host either byintegration into the cellular genome or exist extrachromosomally (e.g.autonomous replicating plasmid with an origin of replication).

Specifically included are shuttle vectors by which is meant a DNAvehicle capable, naturally or by design, of replication in both theactinomycetes and related species and in bacteria and/or eucaryotic(e.g. higher plant, mammalian, yeast or fungal cells).

A vector including nucleic acid according to the present invention neednot include a promoter or other regulatory sequence, particularly if thevector is to be used to introduce the nucleic acid into cells forrecombination into the genome.

However, in a preferred embodiment, the nucleic acid in the vector isunder the control of (operably linked to) an appropriate promoter orother regulatory elements for expression in a host cell such as amicrobial, e.g. bacterial, or plant cell. In the case of genomic DNA,this may contain its own promoter or other regulatory elements and inthe case of CDNA this may be under the control of an appropriatepromoter or other regulatory elements for expression in the host cell.

By “promoter” is meant a sequence of nucleotides from whichtranscription may be initiated of DNA operably linked downstream (i.e.in the 3′ direction on the sense strand of double-stranded DNA).

“Operably linked” means joined as part of the same nucleic acidmolecule, suitably positioned and oriented for transcription to beinitiated from the promoter. DNA operably linked to a promoter is “undertranscriptional initiation regulation” of the promoter.

Generally speaking, those skilled in the art are well able to constructvectors and design protocols for recombinant gene expression. Suitablevectors can be chosen or constructed, containing appropriate regulatorysequences, including promoter sequences, terminator fragments,polyadenylation sequences, enhancer sequences, marker genes and othersequences as appropriate. For further details see, for example,Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrook et al,1989, Cold Spring Harbor Laboratory Press.

Many known techniques and protocols for manipulation of nucleic acid,for example in preparation of nucleic acid constructs, mutagenesis (seeabove), sequencing, introduction of DNA into cells and gene expression,and analysis of proteins, are described in detail in Current Protocolsin Molecular Biology, Second Edition, Ausubel et al. eds., John Wiley &Sons, 1992. The disclosures of Sambrook et al. and Ausubel et al. areincorporated herein by reference. Specific procedures and vectorspreviously used with wide success upon plants are described by Bevan(Nucl. Acids Res. 12, 8711-8721 (1984)) and Guerineau and Mullineaux(1993) (Plant transformation and expression vectors. In: Plant MolecularBiology Labfax (Croy RRD ed) Oxford, BIOS Scientific Publishers, pp121-148).

If desired, selectable genetic markers may be used consisting ofchimaeric genes that confer selectable phenotypes such as resistance toantibiotics such as kanamycin, hygromycin, phosphinotricin,chlorsulfuron, methotrexate, gentamycin, spectinomycin, imidazolinonesand glyphosate.

Thus this aspect of the invention provides a gene construct, preferablya replicable vector, comprising a promoter operatively linked to anucleotide sequence provided by the present invention, such as the afVP1gene shown in Seq ID No 1, a homologue thereof (e.g. taVP1), or anyactive mutant, derivative or allele thereof.

Suitable promoters include the Cauliflower Mosaic Virus 35S (CaMV 35S)gene promoter that is expressed at a high level in virtually all planttissues (Benfey et al, 1990a and 1990b); the cauliflower meri 5 promoterthat is expressed in the vegetative apical meristem as well as severalwell localised positions in the plant body, e.g. inner phloem, flowerprimordia, branching points in root and shoot (Medford, 1992; Medford etal, 1991) and the Arabidopsis thaliana LEAFY promoter that is expressedvery early in flower development (Weigel et al, 1992).

Another strong promoter is the rice actin promoter. Also advantageous inthe present context is the ubiquitin promoter which is expressedstrongly in embryos (see Christenson & Quail (1996) Transgenic Research5: 2133-2218.

Previous work in Arabidopsis has shown that constitutive expression ofABI 3 causes no negative effects on plant growth, and so expression ofafVP 1 throughout a plant (e.g. wheat) may not have negative sideeffects on wheat plant growth.

However the promoter may include one or more sequence motifs or elementsconferring developmental and/or tissue-specific regulatory control ofexpression. Other regulatory sequences may be included, for instance asidentified by mutation or digest assay in an appropriate expressionsystem or by sequence comparison with available information, e.g. usinga computer to search on-line databases.

Thus in another embodiment of this aspect of the present invention,there is provided a gene construct, preferably a replicable vector,comprising an inducible promoter operably linked to a nucleotidesequence provided by the present invention, such as the afVP1 gene, ahomolog from another plant species, e.g. a wheat taVP1, or any mutant,or allele thereof.

The term “inducible” as applied to a promoter is well understood bythose skilled in the art. In essence, expression under the control of aninducible promoter is “switched on” or increased in response to anapplied stimulus. The nature of the stimulus varies between promoters.Some inducible promoters cause little or undetectable levels ofexpression (or no expression) in the absence of the appropriatestimulus. Other inducible promoters cause detectable constitutiveexpression in the absence of the stimulus. Whatever the level ofexpression is in the absence of the stimulus, expression from anyinducible promoter is increased in the presence of the correct stimulus.The preferable situation is where the level of expression increases uponapplication of the relevant stimulus by an amount effective to alter aphenotypic characteristic. Thus an inducible (or “switchable”) promotermay be used which causes a basic level of expression in the absence ofthe stimulus which level is too low to bring about a desired phenotype(and may in fact be zero). Upon application of the stimulus, expressionis increased (or switched on) to a level which brings about the desiredphenotype.

A suitable inducible promoter is the GST-II-27 gene promoter which hasbeen shown to be induced by certain chemical compounds which can beapplied to growing plants. The promoter is functional in bothmonocotyledons and dicotyledons. It can therefore be used to controlgene expression in a variety of genetically modified plants, includingfield crops such as canola, sunflower, tobacco, sugarbeet, cotton;cereals such as wheat, barley, rice, maize, sorghum; fruit such astomatoes, mangoes, peaches, apples, pears, strawberries, bananas, andmelons; and vegetables such as carrot, lettuce, cabbage and onion. TheGST-II-27 promoter is also suitable for use in a variety of tissues,including roots, leaves, stems and reproductive tissues.

Other advantageous promoters may be those which function at particulardevelopmental stages (e.g. embryogenesis)—for instance the Em promoter,or the taVP1 wheat promoter which is discussed hereinafter.

In a fourth aspect the present invention also provides methodscomprising introduction of such a construct into a plant cell and/orinduction of expression of a construct within a plant cell, byapplication of a suitable stimulus, an effective exogenous inducer.

The vectors described above may be introduced into hosts by anyappropriate method e.g. conjugation, mobilisation, transformation,transfection, transduction or electoporation.

However, when introducing a chosen gene construct into a cell, certainconsiderations must be taken into account, well known to those skilledin the art. The nucleic acid to be inserted should be assembled within aconstruct which contains effective regulatory elements which will drivetranscription. There must be available a method of transporting theconstruct into the cell. Once the construct is within the cell membrane,integration into the endogenous chromosomal material either will or willnot occur. Finally, as far as plants are concerned the target cell typemust be such that cells can be regenerated into whole plants (seebelow).

Plants transformed with the DNA segment containing the sequence may beproduced by standard techniques which are already known for the geneticmanipulation of plants. DNA can be transformed into plant cells usingany suitable technology, such as a disarmed Ti-plasmid vector carried byAgrobacterium exploiting its natural gene transfer ability (EP-A-270355,EP-A-0116718, NAR 12(22) 8711-87215 1984), particle or microprojectilebombardment (US 5100792, EP-A-444882, EP-A-434616) microinjection (WO92/09696, WO 94/00583, EP 331083, EP 175966, Green et al. (1987) PlantTissue and Cell Culture, Academic Press), electroporation (EP 290395, WO8706614 Gelvin Debeyser—see attached) other forms of direct DNA uptake(DE 4005152, WO 9012096, US 4684611), liposome mediated DNA uptake (e.g.Freeman et al. Plant Cell Physiol. 29: 1353 (1984)), or the vortexingmethod (e.g. Kindle, PNAS U.S.A. 87: 1228 (1990d) Physical methods forthe transformation of plant cells are reviewed in Oard, 1991, Biotech.Adv. 9: 1-11.

Agrobacterium transformation is widely used by those skilled in the artto transform dicotyledonous species. Recently, there has beensubstantial progress towards the routine production of stable, fertiletransgenic plants in almost all economically relevant monocot plants(Toriyama, et al. (1988) Bio/Technology 6, 1072-1074; Zhang, et al.(1988) Plant Cell Rep. 7, 379-384; Zhang, et al. (1988) Theor Appl Genet76, 835-840; Shimamoto, et al. (1989) Nature 338, 274-276; Datta, et al.(1990) Bio/Technology 8, 736-740; Christou, et al. (1991) Bio/Technology9, 957-962; Peng, et al. (1991) International Rice Research Institute,Manila, Philippines 563-574; Cao, et al. (1992) Plant Cell Rep. 11,585-591; Li, et al. (1993) Plant Cell Rep. 12, 250-255; Rathore, et al.(1993) Plant Molecular Biology 21, 871-884; Fromm, et al. (1990)Bio/Technology 8, 833-839; Gordon-Kamm, et al. (1990) Plant Cell 2,603-618; D'Halluin, et al. (1992) Plant Cell 4, 1495-1505; Walters, etal. (1992) Plant Molecular Biology 18, 189-200; Koziel, et al. (1993)Biotechnology 11, 194-200; Vasil, I. K. (1994) Plant Molecular Biology25, 925-937;

Weeks, et al. (1993) Plant Physiology 102, 1077-1084; Somers, et al.(1992) Bio/Technology 10, 1589-1594; WO92/14828). In particular,Agrobacterium mediated transformation is now emerging also as an highlyefficient alternative transformation method in monocots (Hiei et al.(1994) The Plant Journal 6, 271-282).

Particularly of interest on the present case is the fact that thegeneration of fertile transgenic plants has been achieved in the cerealsrice, maize, wheat, oat, and barley (reviewed in Shimamoto, K. (1994)Current Opinion in Biotechnology 5, 158-162.; Vasil, et al. (1992)Bio/Technology 10, 667-674; Vain et al., 1995, Biotechnology Advances 13(4): 653-671; Vasil, 1996, Nature Biotechnology 14 page 702).

Microprojectile bombardment, electroporation and direct DNA uptake arepreferred where Agrobacterium is inefficient or ineffective.Alternatively, a combination of different techniques may be employed toenhance the efficiency of the transformation process, eg bombardmentwith Agrobacterium coated microparticles (EP-A-486234) ormicroprojectile bombardment to induce wounding followed byco-cultivation with Agrobacterium (EP-A-486233).

Following transformation, a plant may be regenerated, e.g. from singlecells, callus tissue or leaf discs, as is standard in the art. Almostany plant can be entirely regenerated from cells, tissues and organs ofthe plant. Available techniques are reviewed in Vasil et al., CellCulture and Somatic Cell Genetics of Plants, Vol I, II and III,Laboratory Procedures and Their Applications, Academic Press, 1984, andWeissbach and Weissbach, Methods for Plant Molecular Biology, AcademicPress, 1989.

The particular choice of a transformation technology will be determinedby its efficiency to transform certain plant species as well as theexperience and preference of the person practising the invention with aparticular methodology of choice. It will be apparent to the skilledperson that the particular choice of a transformation system tointroduce nucleic acid into plant cells is not essential to or alimitation of the invention, nor is the choice of technique for plantregeneration.

Thus this aspect of the present invention includes a method oftransforming a plant cell involving introduction of a vector comprisingthe afVP1 sequence (or a mutant or derivative thereof) into a plant celland causing or allowing recombination between the vector and the plantcell genome to introduce the sequence of nucleotides into the genome.

In a fifth aspect of the invention, there is disclosed a host cellcontaining nucleic acid or a vector according to the present invention,especially a plant or a microbial cell.

The invention further encompasses a host cell transformed with nucleicacid or a vector according to the present invention, especially a plantor a microbial cell, and most preferably a crop plant e.g. wheat. Withinthe cell, the nucleic acid may be incorporated within the chromosome.There may be more than one heterologous nucleotide sequence per haploidgenome.

Thus in one embodiment of the invention there is provided a plant cellhaving incorporated into its genome nucleic acid, particularlyheterologous nucleic acid, as provided by the present invention, underoperative control of a regulatory sequence for control of expression.The coding sequence may be operably linked to one or more regulatorysequences which may be heterologous or foreign to the gene, such as notnaturally associated with the gene for its expression. The nucleic acidaccording to the invention may be placed under the control of anexternally inducible gene promoter to place expression under the controlof the user.

The term “heterologous” is used broadly in this aspect to indicate thatthe gene/sequence of nucleotides in question have been introduced intosaid cells of the plant or an ancestor thereof, using geneticengineering, i.e. by human intervention. A heterologous gene (e.g.authentic afVP1) may replace an endogenous equivalent gene (e.g. taVP1;i.e. one which normally performs the same or a similar function) or theinserted sequence may be additional to the endogenous gene or othersequence. The heterologous (or exogenous or foreign) nucleic acid may benon-naturally occuring in cells of that type, variety or species. Thusthe heterologous nucleic acid may comprise a coding sequence of orderived from a particular type of plant cell or species or variety ofplant, placed within the context of a plant cell of a different type orspecies or variety of plant. A further possibility is for a nucleic acidsequence to be placed within a cell in which it or a homolog is foundnaturally, but wherein the nucleic acid sequence is linked and/oradjacent to nucleic acid which does not occur naturally within the cell,or cells of that type or species or variety of plant, such as operablylinked to one or more regulatory sequences, such as a promoter sequence,for control of expression.

In the transgenic plant cell (i.e. transgenic for the nucleic acid inquestion) the transgene may be on an extra-genomic vector orincorporated, preferably stably, into the genome.

A plant may be regenerated from one or more transformed plant cellsdescribed above. Such plants form a sixth aspect of the invention.

A plant according to the present invention may be one which does notbreed true in one or more properties. Plant varieties may be excluded,particularly registrable plant varieties according to Plant Breeders'Rights. it is noted that a plant need not be considered a “plantvariety” simply because it contains stably within its genome atransgene, introduced into a cell of the plant or an ancestor thereof.

In addition to a plant, the present invention provides any clone of sucha plant, seed, selfed or hybrid progeny is and descendants, and any partof any of these, such as cuttings, seed. The invention provides anyplant propagule, that is any part which may be used in reproduction orpropagation, sexual or asexual, including cuttings, seed and so on. Alsoencompassed by the invention is a plant which is a sexually or asexuallypropagated off-spring, clone or descendant of such a plant, or any partor propagule of said plant, off-spring, clone or descendant.

Particularly embraced are the seeds or grains of a transformed plant asdescribed above, and also products (e.g. human and animal foodstuffs)derived from or containing such seeds or grains.

In a seventh aspect, the invention provides a method of influencing oraffecting the dormancy characteristics of a plant, preferably theviviparous or PHS phenotype of a plant, including the step of causing orallowing expression of a heterologous nucleic acid sequence, asdiscussed in relation to the first and second aspects of the invention,within cells of the plant.

This aspect particularly provides a method of including expression fromnucleic acid Seq ID No 1 or 2, or a mutant, allele or derivative ofthose sequences, within cells of a plant (thereby producing the encodedpolypeptide), following an earlier step of introduction of the nucleicacid into a cell of the plant or an ancestor thereof. The method mayemploy the vectors of the third aspect.

In the present invention, expression (or over-expression or endogenoussequences) may be achieved by introduction of the nucleotide sequence ina “sense” orientation. Thus, the present invention provides a method ofthe method including causing or allowing expression of the product(polypeptide or nucleic acid transcript) encoded by heterologous nucleicacid according to the invention from that nucleic acid within cells ofthe plant.

The complete sequence corresponding to the coding sequence of afVP1 neednot be used. For example fragments (i.e. active derivatives or mutants)of sufficient length may be used.

The sequence employed may be about 500 nucleotides or less, possiblyabout 400 nucleotides, about 300 nucleotides, about 200 nucleotides, orabout 100 nucleotides. It may be possible to use oligonucleotides ofmuch shorter lengths, 14-23 nucleotides, although longer fragments, andgenerally even longer than about 500 nucleotides are preferable wherepossible, such as longer than about 600 nucleotides, than about 700nucleotides, than about 800 nucleotides, than about 1000 nucleotides ormore.

Down-regulation of expression of a target gene (e.g. a homologidentified in accordance with the second aspect of the invention such astaVP1—Seq ID No 2) may be achieved using anti-sense technology or “senseregulation” (“co-suppression”).

For instance, if it is desired to suppress dormancy (i.e. enhance PHS or‘malting’) then the nucleic acids of the present invention (e.g. taVP1,afVP1, other derivatives) may be used for this purpose in accordancewith standard procedures for anti-sense or sense suppression.

In using anti-sense genes or partial gene sequences to down-regulategene expression, a nucleotide sequence is placed under the control of apromoter in a “reverse orientation” such that transcription yields RNAwhich is complementary to normal mRNA transcribed from the “sense”strand of the target gene. See, for example, Rothstein et al, 1987;Smith et al, (1988) Nature 334, 724-726; Zhang et al, (1992) The PlantCell 4, 1575-1588, English et al., (1996) The Plant Cell 8, 179-188.Antisense technology is also reviewed in Bourque, (1995), Plant Science105, 125-149, and Flavell, (1994) PNAS USA 91, 3490-3496.

An alternative is to use a copy of all or part of the target geneinserted in sense, that is the same, orientation as the target gene, toachieve reduction in expression of the target gene by co-suppression.See, for example, van der Krol et al., (1990) The Plant Cell 2, 291-299;Napoli et al., (1990) The Plant Cell 2, 279-289; Zhang et al., (1992)The Plant Cell 4, 1575-1588, and U. S. Pat. No. 5,231,020. Recent workindicates that foreign (non-endogenous) homologous sequences may beparticularly effective at inducing gene silencing in targeted endogenousgenes. See e.g. Matzke, M. A. and Matzke, A. J. M. (1995), Trends inGenetics, 11: 1-3). This sequence homology may involve promoter regionsor coding regions of the silenced gene (Matzke, M. A. and Matzke, A. J.M. (1993) Annu. Rev. Plant Physiol. Plant Mol. Biol., 44: 53-76,Vaucheret, H. (1993) C. R. Acad. Sci. Paris, 316: 1471-1483, Vaucheret,H. (1994), C. R. Acad. Sci. Paris, 317: 310-323, Baulcombe, D. C. andEnglish, J. J. (1996), Current Opinion In Biotechnology, 7: 173-180,Park, Y-D., et al (1996), Plant J., 9: 183-194.

Thus the sequences of the present invention may have utility when usedin plant species different to those from which they were derived (e.g.barley). In an eight aspect, the present invention also encompasses theexpression product of any of the nucleic acid sequences disclosed,particularly those of the first and second aspects, and methods ofmaking the expression product by expression from encoding nucleic acidtherefore under suitable conditions, which may be in suitable hostcells.

Following expression, the product may be isolated from the expressionsystem (e.g. microbial) and may be used as desired, for instance informulation of a composition including at least one additionalcomponent.

Alternatively (and indeed preferably) the product may perform itsfunction in vivo, in this context the function being to influence thedormancy characteristics of a plant, preferably the viviparous or PHSphenotype of a plant.

In an ninth aspect, purified or semi-purified afVP1 or taVP1 protein, ora fragment, mutant, derivative or other variant thereof, e.g. producedrecombinantly by expression from encoding nucleic acid therefor, may beused to raise antibodies employing techniques which are standard in theart. Antibodies and polypeptides comprising antigen-binding fragments ofantibodies may be used in identifying homologs from other species asdiscussed further below, and also in labelling proteins.

Methods of producing antibodies include immunising a mammal (e.g. human,mouse, rat, rabbit, horse, goat, sheep or monkey) with the protein or afragment thereof. Antibodies may be obtained from immunised animalsusing any of a variety of techniques known in the art, and might bescreened, preferably using binding of antibody to antigen of interest.For instance, Western blotting techniques or immunoprecipitation may beused (Armitage et al, 1992, Nature 357: 80-82). Antibodies may bepolyclonal or monoclonal. As an alternative or supplement to immunisinga mammal, antibodies with appropriate binding specificity may beobtained from a recombinantly produced library of expressedimmunoglobulin variable domains, e.g. using lambda bacteriophage orfilamentous bacteriophage which display functional immunoglobulinbinding domains on their surfaces; for instance see WO92/01047.

Antibodies may have utility in testing for endogenous VP1-analogexpression (especially taVP1 expression in wheat) as part of adormancy/PHS assessment, as is discussed hereinafter.

Antibodies raised to a polypeptide or peptide can be used in theidentification and/or isolation of homologous polypeptides, and thentheir encoding genes.

Thus, the present invention provides a method of identifying orisolating a polypeptide having one or more afVP1 (or taVP1 ) epitopescomprising screening candidate polypeptides with a polypeptidecomprising the antigen-binding domain of an antibody (for example wholeantibody or a fragment thereof) which is able to bind afVP1 (or taVP1 )or a fragment or a derivative thereof or preferably has bindingspecificity for such a polypeptide.

Thus specific binding members such as antibodies and polypeptidescomprising antigen binding domains of antibodies that bind and arepreferably specific for afVP1 or taVP1 polypeptides or mutants orderivatives thereof represent part of the present invention, as do theiruse and methods which employ them.

Candidate polypeptides for screening may for instance be the products ofan expression library created using nucleic acid derived from an plantof interest, or may be the product of a purification process from anatural source. A polypeptide found to bind the antibody may be isolatedand then may be subject to amino acid sequencing. Any suitable techniquemay be used to sequence the polypeptide either wholly or partially (forinstance a fragment of the polypeptide may be sequenced). Amino acidsequence information may be used in obtaining nucleic acid encoding thepolypeptide, for instance by designing one or more oligonucleotides(e.g. a degenerate pool of oligonucleotides) for use as probes orprimers in hybridization to candidate nucleic acid, or by searchingcomputer sequence databases, as discussed further below.

The above description has generally been concerned with the coding partsof the afVP1 gene, and uses therefor. Also embraced within the presentinvention are untranscribed parts of that gene, or the taVP1 gene.

Thus a tenth aspect of the invention is a nucleic acid molecule encodingthe promoter of the afVP1 gene or the taVP1 gene.

The promoter region may be readily identified using a probe or primerbased on Seq ID No 1 or Seq ID no 2, as described in relation to earlieraspects. This can be used in the identification and isolation of apromoter from a genomic library containing DNA derived from a plantsource. Techniques and conditions for such probing are well known in theart as is discussed above. Following probing, promoter activity isassessed using a test transcription system.

“Promoter activity” is used to refer to ability to initiatetranscription. The level of promoter activity is quantifiable forinstance by assessment of the amount of mRNA produced by transcriptionfrom the promoter or by assessment of the amount of protein productproduced by translation of mRNA produced by transcription from thepromoter. The amount of a specific mRNA present in an expression systemmay be determined for example using specific oligonucleotides which areable to hybridise with the mRNA and which are labelled or may be used ina specific amplification reaction such as the polymerase chain reaction.

Use of a reporter gene facilitates determination of promoter activity byreference to protein production. The reporter gene preferably encodes anenzyme which catalyses a reaction which produces a detectable signal,preferably a visually detectable signal, such as a coloured product.Many examples are known, including β-galactosidase and luciferase.β-galactosidase activity may be assayed by production of blue colour onsubstrate, the assay being by eye or by use of a spectro-photometer tomeasure absorbance. Fluorescence, for example that produced as a resultof luciferase activity, may be quantitated using a spectrophotometer.Radioactive assays may be used, for instance using chloramphenicolacetyltransferase, which may also be used in non-radioactive assays. Thepresence and/or amount of gene product resulting from expression fromthe reporter gene may be determined using a molecule able to bind theproduct, such as an antibody or fragment thereof. The binding moleculemay be labelled directly or indirectly using any standard technique.

Those skilled in the art are well aware of a multitude of possiblereporter genes and assay techniques which may be used to determinepromoter activity. Any suitable reporter/assay may be used and it shouldbe appreciated that no particular choice is essential to or a limitationof the present invention.

Also embraced by the present invention is a promoter which is a mutant,derivative, or other homolog of the promoter identified as above. Thesecan be generated or identified in similar manner to the derivativesdiscussed in the second aspect; they will share homology with the taVP1or afVP1 promoters and retain promoter activity.

To find minimal elements or motifs responsible for tissue and/ordevelopmental regulation, restriction enzyme or nucleases may be used todigest a nucleic acid molecule, or mutagenesis may be employed, followedby an appropriate assay (for example using a reporter gene such asluciferase) to determine the sequence required. Nucleic acid comprisingthese elements or motifs forms one part of the present invention.

In an eleventh aspect of the invention there is provided a nucleic acidconstruct, preferably an expression vector, including a promoter regionor fragment, mutant, derivative or other homolog or variant thereof ableto promote transcription as discussed above, operably linked to aheterologous gene, e.g. a coding sequence, which is preferably not thecoding sequence with which the promoter is operably linked in nature.

The above aspects of the invention are concerned generally with methodsand materials which have utility, inter alia, in manipulating PHS and/orother dormancy related traits (secondary and after-ripening) in crops,particularly wheat, by means of transformation.

However the identification of the wheat homolog taVP1 by the presentinventors has also opened up the possibility of improved methods forgenerating plants having desirable characteristics as regards PHS and/orother dormancy related traits. These methods have as their basis theidentification and molecular tagging of the taVP1 gene (using taVP1 cDNAas a probe in genomic southern blots) which has been achieved by thepresent inventors, as described below.

A plant breeding approach to improving the PHS properties of wheat has anumber of advantages over transformation approaches, particularly asregards consumer confidence in the improved products and ease ofregulatory approval.

Essentially, plant breeding is the process of bringing together newcombinations of genes, from different parents, allowing them to reassortinto recombinant genotypes carrying various mixtures of the originalparental genes, then selecting individual progenies which carry geneticcombinations superior to the original parents.

Dormancy is one of a number of characteristics described as“quantitative traits” (i.e. varies over a continuous range as opposed toa trait such as grain colour which is an “all or nothing”, discontinuouscharacter).

Quantitative traits (=QTs) are controlled by several/many genes(situated on the chromosomes at several/many Quantitative TraitLoci=QTLs); and by “environmental” variables including weather effectsand experimental uncertainties of measurement. Breeding for desirableQTs thus demands the ability to effectively discriminate between geneticvariants at a large number of QTLs, conventionally by using statisticaltechniques based on large samples and repeated trials.

Dormancy is a particularly difficult QT for the plant breeder becausethe effects of “error” variables are comparatively large, the traititself is lost gradually during after-ripening, and experimental methodsfor testing dormancy are time/labour/material intensive. Since dormancytests have to be carried out between one harvest and the next sowing(sometimes just a month or so), only limited time is available forempirical testing; for this reason wheat breeders usually defer dormancytesting until the later stages of a breeding programme when only alimited number of “elite” progenies have passed through earlier roundsof selection for other, more easily selectable characters. This has theeffect of reducing the amount of control which the breeder can exerciseover the genetic combinations of dormancy QTLs passing into his newvarieties.

When selecting progeny, rather than use “direct” selection for dormancy(i.e. by measuring the trait itself), in which genetic combinations areunconsciously chosen on the basis of their statistical performance inempirical tests, it is possible to exploit “marker aided” selection,choosing progenies on the basis of discontinuous traits, each of whichis simply controlled by genetic variants at a single (or small number)of genetic locus/loci. The conventional marker for dormancy in wheat isgrain colour; it has been known for many years that red-seeded wheatstend to be more dormant than white-seeded wheats, and that grain colouris determined by dominant “red” versus recessive “white” alleles (genevariants) at three major gene loci. By discarding any white-seededprogenies (detected by visual inspection after steeping grains in diluteaqueous sodium hydroxide) wheat breeders aim to eliminate theundesirable lack of dormancy associated with this character.

Thus one approach to breeding for PHS resistance is disclosed by DereraNF (1989) in “Breeding for pre-harvest sprouting tolerance”. pp111-128in: NF Derera (Ed), “Pre-Harvest Field Sprouting in Cereals. CRC PressInc.” This gives some specific examples of breeding programmes whichhave been undertaken to produce new varieties with improved dormancy,including exploitation of red grain colour, breeding for dormancy inwhite-grained wheats, screening wheat genotypes for use as donors ofdormancy genes in breeding, and a suggested scheme for dormancybreeding. The use of the Hagberg falling number test (to assess theviscosity of ground kernel material under moist and dry conditions) isalso discussed as being a useful measure of sprouting resistance.

Another discussion of PHS is found in Mares D J (1989) “Pre-harvestsprouting damage and sprouting tolerance: assay methods andinstrumentation.” pp129-170 in the same volume as Derera (supra). Thisdisclosed various methods used to select against sprouting. The Hagbergtest, and its approval by various standardisation bodies, is alsodiscussed. It is noted that an absence of sprouting damage (indicativeof low PHS susceptibility) leads to high falling numbers (>400,generally 450-550).

The role of grain colour in dormancy, and its relationship with VP1, hasbeen discussed in number of prior art papers. For instance the poster byWilson (described in the prior art section above) discussed the possibleorthology of the Red grain locus in wheat to the maize VP-1 locus.Similarly Sorrells & Wilson (1997) Crop Science 37: 691-697 discussesthe relationship between maize red pericarp colour (controlled by the Pigene) nd

VP1, and suggests that a (postulated) wheat VP1 homolog may express viaa P homolog.

Unfortunately the association between grain redness and dormancy is nothard and fast: breeders of white-grained wheats (eg. Australia) or amberwheats (eg. durum) must resort to other QTLs, also it has been notedthat red wheats vary widely in dormancy. The most dormant white wheatscan be as dormant as the least dormant reds: within both colour groupsthere is a continuous spectrum of dormancy, the scores of red wheats areshifted to the more dormant end of the spectrum, but there isconsiderable overlap between the two groups. This makes it clear thatgrain colour is not the sole determinant of dormancy.

Thus it can be seen that further markers to assist in the assessment ofPHS resistance and other dormancy-related traits would be beneficial inbreeding improved cultivars.

Ideally such markers should be in as close genetic linkage with the QTLas possible, or even better that it have a direct effect on the QT(reducing or even better eliminating the possibility of recombination).Selecting particular alleles which are known to directly exertparticular phentoypic effects is termed Direct Allele Selection (‘DAS’).Additionally the QTL which is marked should be an important determinantof the QT score. Finally it should also be practicable. Molecularmarkers, which depend on variation (polymorphisms) in the sequences ofbases, are particularly useful in this regard.

The present inventors have now mapped the chromosomal location of thenovel taVp1 genes which they isolated.

Briefly, an RFLP polymorphism was identified between two parents of anF2 mapping population, then the alleles present in each individual ofthe population were determined. These genotypes at the taVp1 locus werethen compared with the genotypes of the same individuals at other,genetically linked loci determined previously (Devos et al 1992). Two ofthe three taVp1 loci were mapped in this way (one on chromosome 3A, oneon 3D); the third locus was detected on chromosome 3B by nullisomicanalysis.

As with the grain colour genes, taVp1 copies reside at three loci on thelong arms of chromosomes 3A, 3B and 3D respectively. The results werecompared to the consensus wheat map of Gale et al 1995 [(M. D. Gale, M.D. Atkinson, C. N. Chinoy, R. L. Harcourt, J. Jia, Q. Y. Li & K. M.Devos. 1995. Genetic maps of hexaploid wheat. pp29-40 in: Proceedings8th International Wheat Genetics Symposium, Eds Z. S. Li & Z. Y. Xin,China Agricultural Scientech Press, Beijing] and homologous taVp1 lociwere assigned to the interval between loci Xwg110 and Xpsr549 on theconsensus map (see FIG. 7).

Interestingly it was found that the taVp1 genes are linked to the R(colour genes) only at a distance of about 25 centiMorgans. This isclear evidence that taVp1 and R genes are different, so that both markersystems can be used for manipulating dormancy, either in concert orindependently. Both markers have direct effects, so loss of effect dueto recombination is not a problem. The linkage interval between the twoloci on each chromosome is large enough to allow a practicably highfrequency of recovery of taVp1/R recombinants should these be required(eg. any existing linkage between a “good” taVp1 allele and a “red”colour allele could realistically be broken for introduction of thetaVp1-based dormancy into white wheats).

Apart from the option for breeding dormant white/amber wheats, taVp1offers advantages over use of the colour marker arising from theavailability of clones of wheat alleles. The problems with the colourmarker are that it cannot be scored until the grain is ripe, and that noinformation is available about which of the R genes is present withoutlengthy and large-scale breeding experiments. Knowledge of the taVp1DNA, RNA, and/or protein sequences allows the identification ofindividual alleles present in a sample of tissue, e.g. DNA from thefirst seedling leaf, or even taken from a seed prior to sowing.

Polymorphisms can be manifest in a number of ways. Structurally theywill alter the characteristics of the DNA to bind probes and primers atparticular sites, or its properties as a substrate for restrictionanalysis. Functionally they may affect the quality or quantity of mRNAor protein product which derives from the DNA. Thus, for instance, thepresence of absence of a lesion in a promoter or other regulatorysequence may be assessed by determining the level of mRNA production bytranscription or the level of polypeptide production by translation fromthe mRNA. The level of mRNA or protein will be affected not only by itsrate of production, but also by its stability and rate of degradation.

Thus the sequence information (nucleic and/or protein product) disclosedherein enables the use of specific amplification, probing or othertechniques, to carry out allele identification and hence germplasmclassification.

“Nucleic acid sequence” in this context embraces the coding sequences,introns, and promoters of the relevant allele, plus alsopost-transcriptional modifications of RNA. Thus tests may be carried outon preparations containing genomic DNA, cDNA and/or mRNA. Testing cDNAor mRNA has the advantage of the complexity of the nucleic acid beingreduced by the absence of intron sequences, but the possibledisadvantage of extra time and effort being required in making thepreparations. RNA may be more difficult to manipulate than DNA becauseof the wide-spread occurrence of RN′ases.

“Protein sequence” in this context covers both the primary structure,plus post-translational protein modifications. Under certaincircumstances the total absence of a detectable protein product will beindicative of alterations in the encoded protein sequence.

Generally the methods may make use of biological samples from one ormore plants or cells (e.g. in a seed)that are suspected to contain thenucleic acid sequences or polypeptide.

The following method are exemplary only. Those skilled in the art willappreciate that other methods which may be devised without burden on thebasis of the information made available by the present inventors alsoform part of the present invention. For instance a number of methods fordetermining the presence and identity of polymorphic molecular markers(in the context of biodiversity analysis) are disclosed by Karp et al(1997) Biotechnology 15: 625-628. Such methods may have analagousutility in carrying out the present invention.

1) At the nucleic acid level, identification may involve hybridisationof a suitable specific oligo- or poly-nucleotide probe, such as afragment of those disclosed herein, or further allelic sequencesestablished using the information disclosed herein. Where the nucleicacid target is double-stranded DNA, hybridisation will generally bepreceded by denaturation to produce single-stranded DNA. Such methodsinclude Southern and Northern hybridisations, which can be used bothqualitatively or quantitively (e.g. to assess mRNA level). A screeningprocedure, chosen from the many available to those skilled in the art,may be used to identify successful hybridisation events and isolatehybridised nucleic acid. For instance, probes may be radioactively,fluorescently or enzymatically labelled.

Preferably the screening is carried out with a variant—orallele-specific probe—this is particular useful for DAS. Such a probecorresponds in sequence to a region of the gene, or its complement,containing a sequence alteration known to be associated with the traitof interest. Under suitably stringent conditions, specific hybridisationof such a probe to test nucleic acid is indicative of the presence ofthe sequence alteration in the test nucleic acid. For efficientscreening purposes, more than one probe may be used on the same testsample.

When screening for particular alleles, the nucleic acid in the samplemay initially be amplified, e.g. using PCR, to increase the amount ofthe analyte as compared to other sequences present in the sample. Thisallows the target sequences to be detected with a high degree ofsensitivity if they are present in the sample. This initial step may beavoided by using highly sensitive array techniques

Approaches which rely on hybridisation between a probe and test nucleicacid and subsequent detection of a mismatch may be employed. Underappropriate conditions (temperature, pH etc.), an oligonucleotide probewill hybridise with a sequence which is not entirely complementary. Thedegree of base-pairing between the two molecules will be sufficient forthem to anneal despite a mis-match. Various approaches are well known inthe art for detecting the presence of a mis-match between two annealingnucleic acid molecules. For instance, RN′ase A cleaves at the site of amis-match. Cleavage can be detected by electrophoresing test nucleicacid to which the relevant probe or probe has annealed and looking forsmaller molecules (i.e. molecules with higher electrophoretic mobility)than the full length probe/test hybrid. Other approaches rely on the useof enzymes such as resolvases or endonucleases. Thus, an oligonucleotideprobe that has the sequence of a region of the normal gene (either senseor anti-sense strand) in which polymorphisms associated with the traitof interest are known to occur may be annealed to test nucleic acid andthe presence or absence of a mis-match determined. Detection of thepresence of a mis-match may indicate the presence in the test nucleicacid of a mutation associated with the trait. On the other hand, anoligonucleotide probe that has the sequence of a region of the geneincluding a mutation associated with disease resistance may be annealedto test nucleic acid and the presence or absence of a mis-matchdetermined. The presence of a mis-match may indicate that the nucleicacid in the test sample has the normal sequence, or a different mutantor allele sequence. In either case, a battery of probes to differentregions of the gene may be employed.

(ii) Allele- or variant- (or even genome-) specific oligonucleotides maysimilarly be used in PCR to specifically amplify particular sequences ifpresent in a test sample. Assessment of whether a PCR band contains agene variant may be carried out in a number of ways familiar to thoseskilled in the art.

The PCR product may for instance be treated in a way that enables one todisplay the mutation or polymorphism on a denaturing polyacrylamide DNAsequencing gel, with specific bands that are linked to the gene variantsbeing selected. It may also be desirable to analyse DNA fragment size,restriction site variation (e.g. CAPS—cleaved amplified polymorphicsites) and so on. Sequence Tagged Site (STS) Polymerase Chain Reaction(PCR) is rapid, specific, and does not require use ofradiosotopes/autoradiography.

By way of Examples, the following primers could be used to distinguishthe certain alleles disclosed in FIGS. 6 and 8. Further primercombinations can be devised without burden by those skilled in the artfor new taVP1 alleles if and when they are identified (for instance byuse of the materials and methods disclosed herein).

Non specific primer (reverse compliment):

Position 2210/clone 10: CGT CAC ATC TGA CCG ATA GC

Primers to differentiate clones 6+9 from 5+10:

6+9 specific: position 1696: CAT CTC AGG TGT GGA GCA TGC

5+10 specific: position 1691: CGG CAC ATC TCA GAT TTT GGC CC

Primers to differentiate clones 5+6 from 9+10:

5+6 specific: position 1432: GCG GCA GCA GGG TGC GAG G

9+10 specific: position 1432: GCG GCA GCA GGT GCA TGC ATG

Further primers which are specific for the A- B- and D-genomes arediscussed in the Examples hereinafter.

(iii) RFLP, hybridized to homologous or heterologous probes based on thesequences disclosed herein. The presence of differences in sequence ofnucleic acid molecules may be detected by means of restriction enzymedigestion, such as in a method of DNA fingerprinting where therestriction pattern produced when one or more restriction enzymes(chosen on the basis of the sequences disclosed herein) are used to cuta sample of nucleic acid is compared with the pattern obtained when asample containing the desired allele or a variant digested with the sameenzyme or enzymes. Amplified Fragment Length Polymorphism (AFLP) can becarried out using primers devised on the basis of the sequencesdisclosed herein. The strength of AFLP is the ability to screen a largenumber of different marker molecules in a single test. Analysis of theproducts can be carried out using e.g. by gel electrophoresis, capillaryelectrophoresis.

(iv) RT-PCR is based on the amplification of RNA, which may bequalitative or quantitative. Essentially this method uses reversetranscriptase to generate a DNA copy of plant mRNA.

(v) Variations in antibody/antigen complexes between plant proteinextracts and antibodies targeted to protein epitopes. This is particularuseful for distinguishing functional from non-functional proteins (e.g.truncated forms, wherein a particular allele contains a premature stopcodon). Thus a sample may be tested for the presence of a bindingpartner for an antibody (or mixture of antibodies), specific for one ormore allelic variants. Binding is assessed by any means commonly knownto those skilled in the art. Where a panel of antibodies is used,different reporting labels may be employed for each antibody so thatbinding of each can be determined.

A specific binding member such as an antibody may be used to isolateand/or purify its binding partner polypeptide from a test sample, toallow for sequence and/or biochemical analysis of the polypeptide todetermine whether it has the sequence and/or properties of the wild-typepolypeptide or a particular mutant, variant or allele thereof. Aminoacid sequence is routine in the art using automated sequencing machines.

Thus the work of the present inventors offers a completely novelapproach for manipulating seed dormancy in breeding programmes,exploiting the taVp1 gene sequence which was previously unknown fordirect selection of alleles controlling a QT for which currentlyavailable techniques are laborious, slow, and inefficient.

Various aspects of the invention will now be discussed in more detail:

In one aspect of the invention there is disclosed a method for assessingthe PHS and/or other dormancy related properties of a wheat plant, themethod comprising use of the molecular marker taVP1 which occurs in theinterval between loci Xwg110 and Xpsr549 on the wheat group 3 consensusmap.

In a further aspect of the invention these is disclosed of producing acultivar comprising the steps of selecting a parent line having desiredPHS and/or other dormancy related properties, breeding with that line,and selecting progeny on the basis of the molecular marker taVP1described above.

Preferably the selection of the parent line(s) and/or progeny is done onthe basis of specific superior alleles (i.e. DAS). This allows precisemanipulation of variation of PHS via selection of progenies withappropriate, desired functional activity of characterised alleles. ThusPHS may be improved by selection for high levels of expression offully-functional alleles, firstly by selecting parents carryingdesirable genomic copies of taVp1 gene(s), then by selecting progenywhich express these alleles strongly at appropriate stages of seeddevelopment and maturation.

The assessment can be on the basis of analysing taVP1 DNA, RNA orprotein as described above, and then correlating the result of theanalysis with the expected PHS phenotype.

Parent plants possessing favoured alleles may be obtained from within anexisting variety genepool, or prepared mutants from within an elitegenepool.

Alternatively desirable alleles or may also be detected and transferredfrom one or more of the many wild or cultivated relatives of the plant,for which established methods are available for the introduction of“alien” variation into the plant genome. The correlation of the PHStrait with the afVP1 sequence greatly facilitates the identification andselection of desirable alleles in exotic germplasm. This is especiallyuseful for species in which genetic variation in cultivated germplasm islimited (e.g. wheat T aestivum—see Chao et al (1989) Theor Appl Genet88:717-721).

In the past, using traditional methods, problems with using exoticgermplasm have included the low frequency of desirable alleles, anddifficulties with linkage drag and polygenic inheritance. Such problemswill be minimised by use of the present invention.

Lines may be produced by breeding from selected lines in accordance withstandard techniques well known to those skilled in the art.

Clearly the PHS phenotype can be manipulated up or down; forapplications in which dormancy is undesirable, e.g. malting, the sameinformation and techniques could be employed to select in the reversedirection, i.e. to fix defective or poorly expressed copies of taVP1.

The demonstration by the present inventors that the afVP1 and seedcolour gene alleles are linked but separate demonstrates that bothmarker systems can be used for manipulating dormancy, either in concertor independently. Thus methods of selection based on taVP1 alone (e.g.in white/amber grained wheats) or both taVP1 and red grain colour (e.g.in red wheats) also form part of the present invention.

Methods in which the taVP1 allele in particular plant or line isassessed and the result of the assessment is correlated directly withexpected PHS phenotype, e.g. for the purposes of timing harvest, form afurther part of the invention.

The taVP1 assessment may also be used to assess pedigree or phylogeneticorigin if desired.

Nucleic acid-based determination of the identity of a particular taVP1allele (e.g. as in the methods described above) may be combined withdetermination of the genotype of the flanking linked genomic DNA andother unlinked genomic DNA using established sets of markers such asRFLPs, microsatellites or SSRS, AFLPS, RAPDs etc. This enables theresearcher or plant breeder to select for not only the presence of thedesirable taVP1 allele but also for individual plant or families ofplants which have the most desirable combinations of linked and unlinkedgenetic background. Such recombinations of desirable material may occuronly rarely within a given segregating breeding population or backcrossprogeny. Direct assay of the taVP1 locus as afforded by the presentinvention allows the researcher to make a stepwise approach to fixing(making homozygous) the desired combination of flanking markers andtaVP1 alleles, by first identifying individuals fixed for one flankingmarker and then identifying progeny fixed on the other side of the locusall the time knowing with confidence that the desirable taVP1 allele isstill present.

The sequence information provided herein also allows the design ofdiagnostic tests and kits for determination of the presence ofparticular taVP1 and afVP1 alleles, in any given plant, cultivar,variety, population, landrace, part of a family or other selection in abreeding programme or other such genotype. A diagnostic test may bebased on determination of the presence or absence of a particular alleleby means of nucleic acid or polypeptide determination.

Plants which are generated (or assessed and or approved) using thetaVP1-allele assessment methods of the present invention form a furtheraspect of the invention. Plants in this context embraces cultivars, andseeds, microspores, protoplasts, cotyledons, zygotes (ovules, pollen)and vegetative parts derived therefrom. It further embraces any clone ofsuch a plant, selfed or hybrid progeny and descendants, and any part ofany of these, such as cuttings and seed. Products made from such plantse.g. milled or malted grains, flour etc. are also embraced by theinvention.

The invention will now be further illustrated with reference to thefollowing non-limiting Figures and Examples. Other embodiments fallingwithin the scope of the invention will occur to those skilled in the artin the light of these.

SEQUENCE ID NOS

Seq ID No 1: (see FIG. 4(a)) “afVP1” CDNA sequence.

Seq ID No 2: (See FIG. 10(a)): “taVP1” CDNA sequence.

FIGURES

FIGS. 1A-1C. Germination behaviour of seed from inbred lines of Avenafatua.

FIGS. 2A-2D. Northern blot analysis of gene expression patterns ofimbibed seed from inbred lines subjected to different environmentalconditions. a. Freshly harvested seed, b. Freshly harvested andafter-ripened seed, c. Seed stored at 4° C. or 24° C. for one year, d.Seed following induction of secondary dormancy (Treated) or Untreated.In each case RNA was extracted from seed following 48 h imbibition. RNAloadings for each sample were 3 μg and 0.5 μg polyA containing RNA perlane. % germination (G%) of seed at the time of RNA extraction isindicated.

FIG. 3. Northern blot analysis shows afVP 1 expression in the dry seedis positively correlated to length of after-ripening time required tobreak dormancy. Germination percentages [G(%)] are shown for four inbredlines 1, 3 and 6 months after harvest. RNA loadings for each sample were3 μg and 0.5 μg polyA containing RNA per lane.

FIG. 4(a): Seq ID No 1, “afVP1” cDNA sequence.

FIG. 4(b): Comparison of afVP 1 predicted protein sequence with other VP1 like transcription factors.

Comparison of the protein sequences from Avena fatua (afVP 1), maize (VP1), rice (osVP 1), bean (ALF 1), Arabidopsis (ABI 3).

FIG. 5. Model for the control of embryo dormancy and afVP 1 RNA levelsby a switch that shows properties of reversibility.

FIGS. 6A-6B. taVP1 allele (clone 4).

FIG. 7: Locations of orthologues in wheat (taVp1), rice (osVP1) andmaize (VP1). From left to right the rice maps are based on Kurata et al(1994a,b) and Quarrie et al (1997), wheat on the concensus map of Galeet al (1995), maize (inverted relative to wheat and rice) on the BNL'95(Anon, 1995) and rice on the map of Causse et al (1994) with additionalmarkers mapped by McCough et al (1996) and the centromeres positionedproximal to cdo920 as demonstrated by Singh et al (1996). Homeologousmarker loci present on two or more maps are joined by dotted lines.Arrows indicate centromeres. Figures to the left of each map denoteintervals in centiMorgans.

FIGS. 8A-8D. Clone 10, representing the taVP1 cDNA sequence. Also shownare further taVP1 alleles (clones 5, 6 and 9) which have been sequencedto various degrees.

FIG. 9. Northern blot analysis of transgenic wheat plants containingafVP1 sequence.

FIG. 10(a): Seq ID No 2, “taVP1” cDNA (clone 10)

FIG. 10(b): clone 2

FIG. 10(c): clone 3

FIG. 10(d): clone 4

FIG. 10(e): clone 5

FIG. 10(f): clone 6

FIG. 10(g): clone 9

FIG. 11. Alignment of sections of six taVP1 clones demonstrating thatthey fall into two groups (group I=4, 5, 10; group II=3, 6, 9).

FIG. 12. Results of PCR using templates specific for the two groups. Theresults using primer set 4 suggest that clone 4 is found on chromosome3A. The results using primer set 9 suggest that clone 9 is found onchromosome 3D.

EXAMPLES Example 1 Analysis of Role of afVP1 in Dormancy andAfter-ripening

Plant Material;

A. fatua inbred lines CS40, AN127, AN51 SH99 used in this study wereobtained from Professor G Simpson (Univ. Of Saskatchewan, Canada). Thelines were derived from single seeds selfed for six generations andgrown in controlled environment rooms (Jana et al. 1988). Inbred lineBampton has been described previously (Hooley et al. 1991, Rushton etal. 1992) derived from single seed selfed for at least 10 generations,inbred line Rewe (Peters 1991) was derived from single seed selfed for 3generations. Mature seeds were obtained from plants grown outdoorsduring the summer each year. Air dry seeds were stored in the dark inconstant environment chambers at 15° C. unless otherwise stated.After-ripening and dormancy levels were monitored every three months bya germination assay.

Germination Assays;

Germination assays were conducted at 22° C. in the dark. Seeds werefirst de-husked, surface-sterilized with 10% (v/v) Parozone (Jeyes Ltd,Norfolk, UK) for 10 minutes, washed with sterile water, and thenincubated embryo-side up on moist sterile glass fibre paper for thetimes indicated in individual figures.

Results:

Six inbred lines of A. fatua were used to study the relationship betweengenetics and environment in the control of embryo dormancy.Dormancy/germination potential of seeds from these lines was assessedover a twelve month period from harvest, following storage at 24° C. Theinbred line CS40 showed an extremely non-dormant phenotype. Dry seedsstored at 24° C. after-ripened within 1 month and embryos germinatedwithin 48 h of imbibition (FIG. 1a). Lines AN51 and SH99 showed higherlevels of primary dormancy than CS40, although this dormancy rapidlydiminished with after-ripening. Line AN127, Rewe and Bampton showed agreater degree of dormancy. Embryos from these lines after-ripenedslowly, taking between 6 months to 1 year to lose dormancy when storedat 24° C. During the period of after-ripening the time taken betweeninitiation of imbibition and germination was much longer in linesshowing dormancy, than in CS40. However this lag time decreased asafter-ripening time increased and dormancy was lost.

Primary dormancy in Bampton was high (FIG. 1a), but after 3 yearsstorage at 24° C. dormancy was lost (indicating that after-ripening wascompleted). However, embryos from seeds stored at 4° C. for the sameperiod were still completely dormant (FIG. 1b), showing that temperatureis an important determinant of the time required for after-ripening.Dormancy of Bampton seeds stored at 4° C. could only be broken followingimbibition at 24° C. by a combination of GA treatment and mechanicalrupture (data not shown).

The effects of conditions that induce secondary dormancy wereinvestigated in three lines, CS40 (non-dormant, ND),

AN127 and Bampton (both dormant, D) following complete after-ripening ofthe embryos (FIG. 1c). Secondary dormancy was induced by immersing seedsin de-gassed water for 70 h at 24° C. in the dark. Embryos were thentested for dormancy potential in the normal germination assay. Finalgermination levels of seeds of the line CS40 were unaffected by theinductive treatment, although germination was slightly delayed. Howeverseeds of AN127 and Bampton were highly susceptible to the treatment,showing reversion to a dormant phenotype following imbibition.

Extraction and Analysis of RNA;

Poly-A containing RNA was extracted from seeds as described previously(Grierson 1992). RNA was size fractionated on 1.5% agaroseMOPS-formaldehyde gels and transferred to a nitrocellulose membrane(Sambrook et al. 1989). Specific mRNAs were detected by hybridisation tomaize VP1 cDNA clones from McCarty et al (1989) The Plant Cell 1,523-532, labelled with α-³²P[dCTP] by random-priming using theStratagene Prime-it II kit according to the manufacturersrecommendations. Hybridisation conditions were 50% formamide, 6×SSPE,5×Denhardts, 0.5% SDS, 100 μg/ml denatured calf thymus DNA, at 42° C.for 16 h. Filters were washed once for 10 min at room temperature in1×SSPE, 0.1% SDS, once for 30 min at room temperature in 1×SSPE, 0.1%SDS, and finally 30 min at 55° C. in 1×SSPE, 0.1% SDS (Sambrook et al.1989). Prior to autoradiography X-ray film was pre-flashed to ensuredetected signals were within the linear range of detection for the film.

Results:

We analysed gene expression patterns in seeds to determine whether theexpression of specific genes was regulated, by genetic background andenvironment, in the same way as dormancy/germination phenotypes in theinbred lines of A. fatua. Two marker genes were chosen that havepreviously been linked to particular developmental states of embryos(AMY 2/1 and Em) (table 1) Other genes analysed were af 10 (expressedthroughout development), and the A. fatua homologue of the maizetranscription factor gene VP 1 (afVP 1).

TABLE 1 Developmental Gene Name: Function: Expression: α-amylase (AMYStarch hydrolysis Germination 2/1) (Avena Specific: fatua)¹ Expressionis repressed by VP 1 in maize.⁵ Em (wheat)² Embryo Maturation:Expression is activated by VP 1 in maize.⁶ af 10 (Avena ? General.fatua)³ afVP 1 (Avena Embryo Embryo Maturation fatua) Transcription inmaize.⁴ Factor⁴ Function and expression patterns of genes used in thisstudy. ¹Hooley et al. 1991, ²Williamson et al. 1985, ³Jones 1996,⁴McCarty et al. 1991, ⁵Hoecker et al. 1995, ⁶Vasil et al. 1995.

We were particularly interested in analysing the expression of the A.fatua VP 1 homologue under the conditions described. The VP 1transcription factor has previously been shown to control embryomaturation in maize, and recent evidence suggests that ABI 3 repressespost-germination developmental processes during embryogenesis (Nambaraet al. 1995). Our initial hypothesis was that the A. fatua homologue ofVP 1 (afVP 1) may regulate processes involved with embryo dormancyfollowing imbibition of the mature seed, by maintaining embryos in thedormant state and inhibiting the dormancy/germination transition. Ifthis was true, then expression of the A. fatua homologue of VP 1 shouldbe linked to the dormant phenotype in imbibed mature seed, and not belimited to embryogenesis. We cloned the A. fatua homologue of VP 1 (afVP1, see section below) and used this cDNA to study expressioncharacteristics of the corresponding RNA. Experiments analyzing theexpression of other genes in this study used homologous probes (AMY 2/1,af 10) and a heterologous probe from wheat (Em). Expression of Emrelated RNAs was studied because this gene has been shown to betranscriptionally activated by VP 1 in maize during embryogenesis(McCarty et al. 1991, Vasil et al. 1995). Transcription of the α-amylasegene AMY 6-4 has been shown to be specifically repressed by VP 1 indeveloping barley seeds, and this gene is a classic marker forgermination (Hoecker et al. 1995).

PolyA-containing RNA was extracted from seeds and analysed by northernblot using radioactively labelled DNA probes. Initially we investigatedthe expression of all the genes in freshly harvested seeds left toimbibe for 48 h (FIG. 2a). Differences in primary dormancy due togenotype were most pronounced at this time (FIG. 1a). AMY-related geneexpression was correlated with lines showing germination (ANS1 andCS40). The af 10 RNA was expressed in all inbred lines, regardless ofphenotype (as were polyubiquitin-related RNAs, data not shown).Expression levels of afEm and afVP 1 RNAs were all increased in linesshowing a dormant phenotype (AN51, AN127, SH99, Bampton and Rewe). Nextwe investigated expression levels of these RNAs in selected lines thathad been allowed to after-ripen (FIG. 2b). There was no difference inexpression of all the different transcripts between fresh andafter-ripened seed of the non dormant line CS40 (ND). There was anincrease in expression of AMY expression in after-ripened AN51 andBampton (although the increase was much less in Bampton), and a largedecrease in expression of the afVP 1 and afEm RNA's in after-ripenedimbibed seed compared to fresh dormant seed.

The influence of temperature of dry seed storage on gene expression wasanalysed using seeds from the dormant inbred line Bampton (FIG. 2c).Seed was stored for 1 year at either 24° C. or 4° C., and then imbibedfor 48 h before RNA extraction. Levels of AMY-related RNA were high inimbibed seeds that had been stored at 24° C. and fully after-ripened,but not in imbibed seed stored at 4° C. that were still dormant. Levelsof RNA corresponding to the afVP 1 and afEm genes were higher in imbibedseeds that had been stored at 4° C. and were still dormant.

We analysed the influence of induction of secondary dormancy on geneexpression using the lines CS40 and Bampton (FIG. 2d). The inductiveconditions had little effect on the final CS40 seed germinationphenotype (FIG. 1c) or on the expression patterns of RNAs analysed inthis inbred line (FIG. 2c). Embryos showed high levels of germination,and high levels of AMY RNA, but low levels of afEm and afVP 1 RNAs. Thesame environmental conditions induced secondary dormancy in Bamptonseeds (FIG. 1c), and associated changes in gene expression. AMY RNA wasdetected at very low levels in treated seeds, whereas afVP 1 and afEmRNAs were present at high levels in treated embryos (D) compared tountreated (ND) embryos.

We analysed whether the depth of dormancy (ie. the length ofafter-ripening time required to break dormancy) shown by seeds from theinbred lines was correlated to the expression levels of afVP 1 in thedry seed. Poly(A)-containing RNA was extracted from dry seeds of linesCS40, AN51 (both show very short after-ripening times, FIG. 1), Bamptonand Rewe (both have long after-ripening requirements), and expression ofafVP 1 RNA was analysed by northern blot (FIG. 3). RNA corresponding toafVP 1 was expressed at similar high levels in the seeds of linesBampton and Rewe, whereas expression of afVP 1 RNA was much lower inseeds from lines CS40 and ANS1. Comparison of the after-ripening periodand levels of afVP 1 expression (FIG. 3) demonstrated a positivecorrelation between the length of time required for after-ripening tooccur and the level of expression of afVP 1 RNA in the dry seed.

Discussion

We have shown that genetic background and environment interact in thedry seed to control the subsequent developmental pathway, and geneexpression programmes, of the embryo following imbibition. This studyshowed a strong correlation between the dormant phenotype and expressionof afVP 1 RNA in both the dry and imbibed seed. The results obtainedsuggest two new features of the biology of VP 1/ABI 3-relatedtranscription factor family. Firstly, the results indicate that aswitching mechanism in the dry seed results in differential expressionof afVP 1 following imbibition. This mechanism results in increasedexpression of afVP 1 in dormant imbibed embryos from mature seeds, andreduced expression during the initiation of germination. Secondly, theresults suggest a new function for VP 1 related transcription factorsduring dormancy in addition to already described functions inembryogenesis, as regulators of post-imbibition dormancy-relatedprocesses.

The different inbred lines used in this study showed different degreesof primary dormancy and rates of after-ripening. In all cases, degree ofdormancy and rate of after-ripening were positively correlated,indicating that the two processes were related. Temperature of storagealso influenced after-ripening, a low temperature increasing theafter-ripening period. This suggests that temperature effects themechanism regulating after-ripening. Dormancy could be re-introduced(secondary dormancy) into embryos by a specific treatment, but only toembryos of those lines that originally showed primary dormancy. Theseresults show that dormancy can only be is re-introduced into embryosthat have the capacity for primary dormancy, ie. those that have agenotype conferring dormancy. The results also suggest that primary andsecondary embryo dormancy are both controlled by the same genetic lociresponsible for the maintenance of primary dormancy. Those embryosshowing primary dormancy also had the capacity for secondary dormancy.These results suggest that the switch mechanism operating in matureseeds may show some features of reversibility (FIG. 5). This modelpredicts that the switch determines the degree of primary dormancy, butcan also be reactivated (reversed) by environmental conditions to inducesecondary dormancy following loss of primary dormancy. In mature seeds,the switch controls the developmental decision of whether the seed willbecome dormant or germinate on imbibition, and of gene expressionprogrammes in imbibed embryos (see below). A bistable switch haspreviously been postulated to control dormancy (Trewavas 1987) byinteractions between kinases and phosphatases. The product of the ABI 1gene from Arabidopsis (mutant abi 1 effects include disruption ofdormancy) is a calcium-modulated phosphatase and could possibly fulfilthis role (Leung et al. 1994).

Our analysis of gene expression following imbibition of seeds shows thatdormant and germinating embryos carry out very different expressionprogrammes. Other studies have shown that several genes of unknownfunction are up regulated in dormant embryos (Johnson et al. 1995, Liand Foley 1995). We have shown that expression of the afVP 1 gene waspositively correlated to the dormant phenotype under all the conditionswe tested, in both the dry and imbibed seed. In addition, afEm RNAshowed a similar pattern of regulation. These results suggest thereforethat these RNA's are regulated by developmental decisions that occur inthe mature seed (FIG. 5). In particular it is noteworthy that afVP 1expression in the dry seed was shown to be correlated with the depth ofdormancy shown by inbred lines. That is, those lines that take longestto after-ripen contained the most afVP 1 RNA the dry seed, whereas thoselines that after-ripen very quickly contained very low levels (FIG. 3).Thus the amount of afVP 1 RNA in the seed at the very onset ofimbibition may determine to some degree the dormancy/germination fate ofthe seed, and this RNA is laid down in the seed during the final stagesof embryo maturation.

The observation of positive correlation between afVP 1 expression in thedry seed and after-ripening requirement shows that this gene could beused as a molecular marker for dormancy potential/after-ripening time.

High levels of expression in the dry seed would indicate a higher degreeof dormancy/ longer after-ripening requirement. The relationship betweenafVP 1 expression in dry and imbibed seed, and embryo genotype indicatesthat this-gene may represent previously described A. fatua loci L1 or L2(Jana et al. 1979, Jana et al. 1988), which influence the degree ofdormancy.

Previous work has demonstrated that VP 1/ABI 3 act in the maturationstage of embryogenesis (Hattori et al., 1992, McCarty et al. 1991,Nambara et al. 1995, Parcy et al. 1994), and do not function followingseed desiccation, (for example ABI 3 RNA expression is reduced rapidlyon imbibition of mature Arabidopsis seed [Parcy et al. 1994]). Theup-regulation of the afVP 1 RNA in dormant A. fatua embryos suggeststhat the encoded protein may play a role in maintaining the dormantstate. Our results suggest that one function for afVP 1 in dormantembryos could be the transcriptional activation of the A. fatua Em gene.Maize VP 1 has previously been shown to regulate Em during embryomaturation by activation of Em transcription through specificcis-elements (Vasil et al. 1995), and afVP 1 could function in a similarway. It would be interesting to analyse the relationship between genesregulated by afVP 1 in dormant embryos and during embryogenesis todefine if this transcription factor functions in a similar or differentway in these two different developmental states. Recent results showthat ABI 3 is also involved in repressing post-germination developmentalprocesses during embryogenesis (Nambara et al. 1995). Another functionof afVP 1 in imbibed dormant seeds may therefore be the inhibition ofgermination-related processes (and germination-related gene expressionsuch as α-amylase).

Example 2 Isolation of afVp1—cDNA Library Construction and Manipulationof Nucleic Acids

cDNA library construction was carried out as previously described(Holdsworth et al. 1992) using poly-A containing RNA from mature embryosof inbred line Bampton. Oligo dT primed cDNA was ligated into the vectorλ-MOSSlox (Palazzolo et al. 1990), and screened according to themanufacturers recommendations (Amersham International plc, UK).Five-prime RACE (rapid amplification of CDNA ends) was carried out usingthe Marathon cDNA amplification kit (Clontech Laboratories Inc, CA,USA). RACE-PCR was primed with a synthetic oligonucleotide correspondingto positions 878-898 of the full-length afVP 1 cDNA. Ligation andsub-cloning of DNA fragments were carried out as described in Sambrooket al. (1989). Sequencing of cloned RACE-PCR amplification products wasperformed manually by the dideoxy chain termination method (Sanger etal. 1977). Sequencing of λMOSSlox subclones was done using a DuPontGenesis 2000 Automated Sequencer (Univ. of Bristol Molecular RecognitionCentre, UK). DNA sequence analysis was carried out using the MacVector™and AssemblyLine™ programmes (Oxford Molecular Group plc, UK) and GCG8(University of Wisconsin Genetics Computer Group version 8 [GeneticsComputer Group 1994]).

Results

The DNA sequence corresponding to the afVP 1 RNA was obtained by acombination of cDNA cloning and 5′RACE (rapid amplification of cDNAends). The combined length predicted from these sequences is 2338 basesfor the full-length RNA, which is similar to the size observed inNorthern Blot analysis of afVP 1.

The predicted protein is 662 amino acids long, smaller than all other VP1/ ABI 3 homologues. The protein sequence of afVP 1 derived from thecDNA was compared to predicted protein sequences of homologues frommaize (VP 1, McCarty et al. 1991), rice (osVP 1, Hattori et al. 1994),Arabidopsis (ABI 3, Giruadat et al. 1992) and Phaseolus vulgaris (ALF 1,Bob et al. 1995) using the GCG8 programme Pileup (Genetics ComputerGroup 1994).

Analysis of the predicted protein sequence of afVP 1 shows that it ishighly similar to other VP 1/ABI 3-related proteins, particularly in thefour regions previously shown to be highly conserved. These regions maybe involved in protein structure or be conserved functional domains. Theregion between amino acids 386 and 407, BR2 (Basic Region 2), in VP 1has previously been shown to interact in-vitro with several differentclasses of transcription factor, including EmBP 1, previously shown tobe involved in the regulation of the Em gene (Hill et al. 1996). Thepredicted protein sequence of afVP 1 shows high homology with VP 1/ABI 3in this region, suggesting a similar functional role in A. fatua. Thein-vivo importance of the BR2 region for the function of thistranscription factor family is indicated by the observation that BR 2occurs at position 439-475 in ABI 3, and the severe allele abi 3-4contains a mutation that converts Gln 417 to a premature stop codon(Giraudat et al. 1992, FIG. 3). In addition, the fourth (and largest)highly conserved region lies downstream of the abi 3-4 premature stopcodon, suggesting an important role for this region also, although nofunction has yet been proposed for this region. Other regions of theprotein, including those shown in maize to regulate Em transcription andAMY repression show low homology, and may be responsible for differentfunctions of the proteins or differences in efficiency of interactionwith other proteins.

Example 3 Cloning taVP 1 Using afVP 1

Plant Material;

Wheat variety Soleil was obtained from Dr John Flintham (John InnesCentre, Norwich, UK). This variety was chosen because it has a highresistance to PHS. Mature seeds were obtained from plants grown outdoorsduring the summer. Air dry seeds were stored in the dark at −20° C. tomaintain dormancy levels prior to RNA extractions.

Germination Assays

These were conducted as described for afVP1.

Extraction and Analysis of RNA From Wheat Seeds

Poly-A containing RNA was extracted from seeds as described previously(Rushton et al 1995). Otherwise the process was as for afVP1.

cDNA Library Construction Screening and Manipulation of Nucleic Acids

cDNA library construction was carried out a previously described(Holdsworth et al 1992) using poly-A containing RNA from mature embryosof the wheat variety Soleil. 500 Soleil seeds (harvested 1996) wereimbibed at 20° C. for 8 hours. Embryos were dissected out and poly(A)RNAextracted (approximately 65 μg). Northern analysis using a fragment ofafVP 1 (bases 600-1892) as a probe confirmed the presence of wheathomologues in the RNA preparation. 5 μg poly(A)RNA was used to constructthe cDNA library (Amersham plc cDNA synthesis kit). Oligo dT primed cDNAwas ligated into the vector λXAP II (Stratagene). 10⁶ plaque-formingunits from the primary library were amplified to obtain an amplifiedlibrary with final titre of 10⁷ pfu/ml. This library was screened usingthe hybridisation and washing conditions described above for Northernanalysis. Wheat VP 1 homologues were identified using as a probe eithera fragment of the afVP 1 cDNA from the 5′ end (basis 1-892 of afVP 1),or a fragment from the middle of afVP 1 (bases 600-1892). A total of 8cDNA clones were purified and characterised, all showing specifichybridisation of the afVP 1 cDNA.

Sequencing of cloned cDNA products was performed manually by the dideoxychain termination method (Sanger et al 1997), or for the longest cDNAclone (Clone 10, 2.3 kbp) via contracting out (Oswel, University ofSouthampton). DNA sequence analysis was carried out using the MacVector™and AssemblyLine™ programmes (Oxford Molecular Group plc, UK) and GCG8(University of Wisconsin Genetics Computer Group version 8 [GeneticsComputer Group 1994]). The wheat cDNA clones shared 81% DNA sequenceidentity with the afVP 1 clone.

Example 4 Method for Reducing PHS in Wheat by use of afVP1 Sequence

Wheat transformation is conveniently carried out according to Barcelo &Lazzeri (1995) in Methods in Molecular Biology, Vol 49. Chapter 9, pp113-124; Ed H Jones, Humana Press, Totowa, New Jersey. This employsmicroprojectile bombardment of immature inflorescence and scutellumtissues; the content of this paper is indicative of the ability of thoseskilled in the art to perform wheat transformation without burden. Othercommon methods for transforming wheat are discussed by Christou inTrends in Plant Science (1996)1, 12: 423-431, and by Chang et al in U.S. Pat. No. 5,610,042. For the avoidance of doubt any content of thesedocuments not forming part of the common general knowledge is hereinincorporated by reference. Briefly, the full-length afVP 1 cDNA (or atruncated derivative) is cloned into a wheat transformation vectordownstream of the rice actin promoter, which confers constitutiveexpression of afVP 1, or the ubiquitin promoter. Immature embryos orinflorescences are bombarded with gold microcarriers coated with plasmidDNA. Explants are cultured, selected and plants are regenerated.Transgene expression is then assayed e.g. using a GUS marker.

More specifically transgenic wheat containing sense and antisense afVP1constructs was produced and analysed as follows:

a. Description of afVP1 Constructions:

Sense and antisense versions of afVP1 were cloned into the planttransformation/expression vector pUPLN, that contains the ubiquitinpromoter upstream of a multiple cloning region (MCR). This planttransformation vector was constructed in the laboratory of Dr. PaulLazzeri (IACR-Rothamsted). It contains the Ubiquitin promoter and firstintron and exon, and the NOS terminator DNA sequence from the plamidpAHC17 (Christensen and Quail, Transgenic Research 5, 213-218; 1996).These DNA sequences were introduced into the plamid pSP72 to createPUPLN.

A Notl DNA fragment containing only the complete afVP1 CDNA wasintroduced into pUPLN at the Not I site within the MCR. Subclone's wereidentified as containing the afVP 1 in the “sense” of “antisense”orientation with respect to the ubiquitin promoter, by digestion ofplamid DNA with Spel (that distinguishes afVP 1 insert orientationbecause both the afVP 1 cDNA and pUPLN contain a single site each).

b. Transformation of Wheat

This was carried out as described above. pUPLN plamid DNA containingafVP 1 in either sense of antisense orientation were separatelytransformed into wheat (cultivars used were; Cadenza, Canon, Riband, lmpand Avans, information is shown for Cadenza and Canon).

C. Identification of Transgenic Plants:

i) Using PCR to Show Wheat Plants Contain afVP 1 DNA Sequence:

PCR was carried out as already described.

Oligonucleotide's specific to afVP 1 were used with wheat genomic DNAderived from putative transgenic plants to amplify a portion of the afVP1 CDNA within the PUPLN plamid integrated into wheat genomic DNA.

The oligonucleotides were:

(5′: 63967: bases 1410-1429 of EMBL deposited afVP 1 sequence):

5′ CAA CTC ATG GTC CCG AAT CC 3′

(5′: afVP1 EPRIME: bases 2285-2302 of EMBL deposited afVP 1 sequence):

5′ GCT TGT TAG ACG AAT TGA C 3′

The results of this experiment demonstrated that transgenic plants havebeen identified that separately contain copies of sense and antisenseafVP 1 cDNA.

ii) Northern Analysis of Transgenic Antisense Plants:

Total RNA was extracted from leaf material using a Qiagen™ kit.

N-blot production and analysis carried out as described in Sambrook etal (1989, supra). The probe used was a radiolabelled an afVP 1, 1.4 KbpNot/Hind III DNA fragment, containing 1.4 kbp at the 3 prime end of thecDNA.

The results of this experiment are shown in FIG. 9. This shows that thetransgenic plants analysed express antisense afVP 1 in leaf material toa very high level. One specific hybridising transcript is detected inleaf total RNA from transgenic line #33 (within cultivar Cadenza).Control RNA derived from untransformed leaf material of the samecultivar did not show hybridisation to the afVP 1 probe.

Example 5 Mapping taVP1 and OsVP1

Mapping in Wheat

An afVP1 CDNA clone (‘lars10’—taVP1 ) was used to identify thechromosomal location of taVp1 loci in hexaploid wheat and identify arestriction fragment length polymorphism (RFLP) which was used forgenetic mapping. Using nullisomic-tetrasomic lines of the wheat cultivar‘Chinese Spring’ in genomic Southern analysis, the presence of taVp1homologs on each group 3 chromosome was established. Two RFLPs wereidentified between the wheat cultivars ‘Chinese Spring’ and ‘Synthetic’in an EcoR1 DNA digest. This enabled taVp1 to be mapped on chromosomes3A and 3D using the F₂ population from ‘ChineseSpring’×‘Synthetic’ crossof Devos et al (1992).

The 3B orthologue was not polymorphic in digests with EcoRl, EcoRV, DraIor HindIII and was not mapped. Mapping data were incorporated into theexisting linkage map for this cross using MAPMAKER version 3.0. Onchromosome 3A, Xlars10 (taVp1) is positioned distal to marker Xpsr549 by12.1 cM and proximal to marker Xabg389 by 3.9 cM. The 3D homoeolocus isdistal by 12.1 cM to Xpsr 170 and proximal to Xpsr 1067 by 5.5 cM. Thesetwo gene locations are consistent with the location shown in FIG. 7, thethe interval Xpsr549—Kwg110 on the wheat group 3 consensus map of Galeet al. (1995), about 30 cM from the centromere. Xlars10 (taVp1) showsclear recombination with R loci which map about 60 cM from thecentromere. Thus, although Xlars10 (taVp1) and R are linked, they areclearly distinct genes.

Thus the results show that both wheat carries a Vp1 orthologue in loosegenetic linkage with previously mapped genes controlling seedcoatpigments (R loci in wheat). It is proposed that this genetic separationbetween the two loci reflects separate roles for taVp1 and Rrespectively in zygotic and maternal dormancy mechanisms. To date no QTLmapping the proximity of Xlars10 had been detected in wheat (Anderson etal 1993). QTL for malting quality that map to the long arm of chromosome3 in barley vary in position between genetic crosses and differentenvironments, and it is not clear whether these QTL reflect effects ondormancy (compare for example, Hayes et al. 1993 c.f Oberthur et al.1995).

Assigment of VP1 cDNA Clones to the A and D Genomes of Wheat

Background

An alignment of sequences from six Vp1 cDNA clones demonstrated that theclones fall into two main groups characterised by single base pairdifferences at a number of places along the sequence, illustrated inFIG. 11 for a section of cDNA (group 1: clones 4, 5 and 10; group 2:clones 3, 6 and 9). Each group appears to represent a single genomiccopy gene (one set from a gene on a chromosome 3A, the other on 3D). Thedifferences between the clones on each family may therefore have arisenby post-transcriptional modifications (e.g. differential splicing toform mature mRNA). This suggests that there may be advantages in taggingtaVP1 mRNA and/or polypeptides (rather than genomic alleles) to assesstaVP1 function (and hence PHS or other dormancy traits)

Methods

PCR primers were designed to specifically amplify each sequence groupfrom genomic DNA. FIG. 11 illustrates the location of the 3′ end of theforward primers, designed to coincide with the base pair differencesexisting between each group:

primer 4 forward: 5′ AATATCTGATACGCGGCGTGAAGGTG3′

primer 9 forward: 5′ AGGATCTAGCCAAGCACAAGAATGG3′

The 3′ end of the reverse primers were designed in a similar way:

primer 4 reverse: 5′GCCCATATGAACTCGATCGATTGAC 3′

primer 9 reverse: 5′GTTGTCCATATGAACTCGATCGATTC 3′

The reaction conditions are described below:

1X Taq buffer (Boehringer)

1.2 mM MgCl₂

200 μM of dGTP, DATP, dUTP and dCTP

0.4 μM of each primer

1.5 U/100 μl Taq polymerase (Boehringer)

50 ng genomic DNA

The total reaction volume was 50 μl. The PCR cycles were:

Five cycles of:

94° C. 1 minute

70° C. 1 minute

72° C. 1 minute

Thirty cycles of:

94° C. 45 seconds

65° C. 1 minute

72° C. 1 minute

Results

The result of the PCR on various templates is shown in FIG. 12. PlasmidDNA from clones four (4) and nine (9) represent the two sequence groupsand act as control templates; primer set 4 is specific to one group andprimer set nine is specific to the other. For genomic DNA from anullisomic-tetrasomic line of Chinese Spring in which the 3A chromosomeis absent (N3A/T3D), amplification does not occur. However, when the 3Bchromosome (in N3B/T3A lines) or 3D chromosome (in N3D/T3A lines) isabsent amplification can still occur. This demonstrates that clone 4represents the gene located on chromosome 3A.

For the primer set designed to the other sequence group, amplificationoccurs using DNA from nulli-tetra lines in which the 3A and 3Bchromosomes are missing but amplification is abolished in lines missing3D. This demonstrates that clone 9 represents the gene located onchromosome 3D.

Genomic clones for all three genome copies have been identified in aSoleil genomic library. Genome-specific primers will be useful fortargetting each copy of Vp1 and studying correlations of particular Vp1alleles with dormancy/preharvest sprouting. They may also haveparticular utility in assessing introgression into specific wheatgenomes, for instance from wild relatives.

Example 6 Use of the taVP1 as a Marker for Dormancy in BreedingProgrammes

Superior alleles of taVp1 may be identified by cloning (e.g. usingprobes/primers based on the sequences disclosed herein e.g. in FIG. 10)and sequencing alleles from a variety of lines, and correlating thesequence with the PHS properties of those plants. PCR primers which arespecific for “superior” alleles can then be used to select preferredgenotypes. This allows early-generation selection against PHS using arapid, small-scale method, in contrast with current practise of delayingselection until late generations and using extremely cumbersome andrather unreliable empirical dormancy tests.

Dormancy may be improved by selection for high levels of expression offully-functional alleles, firstly by selecting parents carryingdesirable genomic copies of taVp1 gene(s), then by selecting progenywhich express these alleles strongly at appropriate stages of seeddevelopment and maturation.

Desirable alleles may be obtained from within the existent wheat(Triticum aestivum) genepool, but may also be detected and transferredfrom one or more of the many wild or cultivated relatives of wheat, forwhich established methods are available for the introduction of “alien”variation into the hexaploid. The recombining of wheat/alien chromosomesis a standard technique, see e.g. M. D. Gale & T. E. Miller. 1987. Theintroduction of alien genetic variation into wheat. ppl73-210 in: WheatBreeding Its Scientific Basis. Ed. F. G. H. Lupton. Chapman & Hall.Wheats carrying such alleles could then be crossed into a commercialbreeding programme and varieties resistant to PHS due to the presence ofsuperior alleles could be identified eg. by PCR using allele-specificprimers.

For applications in which dormancy is undesirable, eg. grain forbrewing, the same information and techniques could be employed to selectin the reverse direction, i.e. to fix defective or poorly expressedcopies of taVp1 (malting wheat).

Example 7 Use of the taVP1 for Predicting the Susceptibility to PHS

A taVP1 based test for predicting the onset of PHS in commercial cropswould enable farmers to prioritise harvesting of high-quality varieties.Present attempts to do this only use weather data (P. S. Kettlewell, G.D. Lunn, B. J. Major, R. K. Scott, P. Gate & F. Couvreur, 1995. Apossible scheme for pre-harvest prediction of Hagberg Falling Number andsprouting of wheat in the U.K. and France. p35-41 in: Pre-HarvestSprouting in Cereals 1995. Eds. K. Noda & D. J. Mares. Centre forAcademic Societies Japan). The level of effective taVp1 activity in theseed may be measured e.g. using a PCR-based or antibody assays in orderto predict susceptibility to PHS.

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Hattori T, Terada T, Hamasuna S T (1994) Sequence and functionalanalyses of the rice gene homologous to the maize Vp1. Plant Mol. Biol.24:805-810.

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Quarrie (1997) PMB submitted Saghai Maroof M A, Yang G P, Biyashev R M,Maughan P J, Zhang Q (1996) Analysis of the barley and rice genomes bycomparative RFLP linkage mapping. Theor Appl Genet 92:541-551.

Van Deynze A E, Nelson J C, O'Donoughue L S, Ahn S N, Siripoonwiwat W,Harrington S E, Yglesias E S, Braga D P, McCouch S R, Sorrells M E(1995a) Comparative mapping in grasses. Oat relationships. Mol. Gen.Genet. 249:349-356. Van Deynze A E, Nelson J C, Yglesias E S, HarringtonS E, Braga D P, McCouch S R, Sorrells M E (1995b) Comparative mapping ingrasses. Wheat relationships. Mol Gen Genet 248:744-754.

Further References

Anderson et al (1993): Crop Sci 33:453-459

Hayes et al (1993): Barley Genet Newsl 23:98-142

Oberthur et al (1995): J Quant Trait Loci (8pp, onlinehttp://probe.nalusda.gov:8000/otherdocs/jqtll995-05/dormancy.html)

34 1 2324 DNA Avena fatua 1 gcacacccct tcttccctcc ttccctccct ccctcctcctgccttccctt tgcatggacg 60 cctccgccgg ctcctcgccg ccgccgcact cgcaggagaacccgcccaag cacggtggag 120 gccgcgggaa gcgtgcgggg gagatccgga agggagaggcggccacggcg gatgacttta 180 tgttcgcgga agataccttc ccgtccctcc cggatttcccttgcctctcc tcccgttcaa 240 gctccacctt ctcctcctca tcctcctcca actcatccagcacccacgcc gccgcgggac 300 gcggcgtggc cgttgtcgcg gacgcccgaa ggcgcctcggggagccctcc gatcctgctg 360 ccgcggggga cgatgacgtg ctcgacgaca tcgacgagctgctcaactct gccacgctct 420 ccgactccat gccctgggag gacgagccgc tcttccccgacgacgttggc atgatgatag 480 aggacgccat ctcccaccag ccgcccgcca cgggccaccgcggagccagg aacgctgcat 540 catcggaggc ggctgctggt ggtggtggac aggattcctcgtcggcggac gacctgccgc 600 ggttcttcat ggagtggctg acgaacaacc gcgactgcatctccgccgag gacctccgca 660 gcatccgcct ccgccgctcg accatcgagg ccgcggcggcgcggctcggt ggagggcggc 720 agggcaccat gcagctgctc aagctcatcc tcacatgggtgcagaaccac catctgcaga 780 agaagcgcgc ccgcgtcgac gacgagctcc ccagccccggcgcaaacccg ggttacgagt 840 tccccgcgga gacagttgcc cccgccacat cctggctcatgccctaccaa caagcttatg 900 gaagagaggc gatctacccg aacgccgccg ccaccgggcagtacccattc cagcagggcg 960 gcagcacgag cagcgtggtg gtgagcagcc agccgttctccccgccggcg ccggtggccg 1020 acatgcaggc ggcgaacatg ccctggccgc agcagtacgcggcgttcccc ggcgctgcgc 1080 catacccgat gccgccgccg cagccgttgg cggcggccggattcggcgtg tgcccgcagc 1140 ccttggccgg ggtgaagccg tcggcgagca aggaggcccggaagaagcgt atggcgaggc 1200 agcgccgcct ctcctgcctg cagcatcagc ggagccagcagctgaatctg ggccagatcc 1260 agaacgccat gatccatccg cagcaggagg tgccgttctctccccgctcc gcgcactcgg 1320 tgcctgtctc accgccgtcg cccggcggct ggtgcgggctctggccgccg ccctccgtcc 1380 aagtccaggg ccagggccaa ctcatggtcc cgaatccgctgtcgacaaag cccagttcct 1440 cctcgaggca gaaggcgcag aaaccctcgc cggacgcaggagcaagaccg ccgtcgtccg 1500 gcgcgcagca gggtgcgaag ccgggggcgg acaagaatctgcggtttctg ctgcagaagg 1560 tgctgaagca gagcgacgtc ggcgccctcg gccgcatcgtgctccccaaa gaagcggaga 1620 cgcacctgcc ggagctcaag acgagggacg gcatctccatccccatggag gacatcggca 1680 cctctcgggt ctggagcatg cggtaccggt tttggcctaacaacaagagc agaatgtatc 1740 tccttgagaa cactggggac tttgttcgct caaacgagctgcaggagggc gacttcatcg 1800 tgatttactc agatgtcaag tcgggcaaat atctgatacgtggtgtgaag gtaagaccgc 1860 cgcaggatct agcgaagcag aagcatggca gtctagagaaaggcagcacc tcagatgcga 1920 tgccctgcgc tgaagacggt ggcgccgagg caggcggctgcaaggggaag tctccgcacg 1980 gcgttaggcg gtctcgccag gaggctgcgt ccatgaaccagatggcggtg agcatctgaa 2040 agaacagccc tagacgatcc accattgaag acttagctagctcgtgtata catgatgttg 2100 atgatcaaat cgatctctgg caccgttgta ttatccgtagtactctagcc ctagggatgg 2160 ttatatatta aagtagctat cagtccgatg tgacgactaaagaatgcatg gtttggttcg 2220 ttaaaaccct gtaaccctgt acatgcatga acataataacttatttgtcg tgtcaattcg 2280 tctaacaagc agactagttc ctgccgtaaa aaaaaaaaaaaaaa 2324 2 2332 DNA Triticum aestivum 2 ggcacgagga cgacttcatgttcgcgcacg ataccttccc ggccctcccg gacttccctt 60 gcctctcctc gccgtcgagctccaccttct cctcctcgtc gtcttccaac tcctccagcg 120 ccttcacccg cgccgtgggggcaggcgggc gcgggggcga gagtgcgcgc ggcgagccgt 180 ccgagcctgc cgcggccggggacgggatgg acgacctctc cgacatcgac cacctgctcg 240 acttcgcatc catcaacgaggacgtccctt gggacgacga gccgctcttc cccgacgtcg 300 ggatgatgct ggaggacgtcatctccgagc agcagcagtt gcaacctccg gcgggccacg 360 gcacggccgg gagaacggcgtcgcatgcgg ctgctggtgg aggagaggat gccttcatgg 420 gtggcggcgg cacggggagcgcggcggacg acctgccgcg cttcttcatg gagtggctca 480 agaacaaccg cgactgcatctcggccgagg acctccgcag catccgcctc cgtcgatcca 540 ccatcgaggc cgcggccgcgcgcctcggtg gggggcgcca gggcaccatg cagctgctca 600 agctcatcct cacctgggtgcagaaccacc acctgcagaa gaagcgcccc cgcgtcggcg 660 ccatggatca ggaggcgctgccggcaggag gccagctccc tagccccggc gcaaaccccg 720 gctacgaatt ccccgcggagacgggtgccg ccgctgccac atcttggatt ccctaccagg 780 ccttctcgcc aactggatcctacggcggcg aggcgatcta cccgttccag cagggctgca 840 gcacgagcag cgtgggcgtgagcagccagc cgttctcccc gccggcggcg cccgacatgc 900 acgccggggc ctggccgctgcagtacgcgg cgttcgtccc agctggggcc acatccgcag 960 gcactcaaac atacccgatgccgccgccgg gggccgtgcc gcagccgttc gcggcccccg 1020 gattcgccgg gcagttcccgcagcggatgg agccggcggc gaccagggag gcccggaaga 1080 agaggatggc gaggcagcggcgcctgtcgt gcctgcagca gcagcggagc cagcagctga 1140 atctgagcca gatccaaaccggcggcttcc ctcaagagcc atccccccgc gcggcgcact 1200 cggcgccggt cacgccgccgtcgtctggct ggggaggcct ctggacgcaa caagccgtcc 1260 agagccagcc ccatggccagctcatggtcc aggtcccgaa tccgctgtcg acgaagtcca 1320 attcctcaag gcagaagcagcaaaaaccct cgccggacgc agcagcgagg ccgccctccg 1380 gcggcgccgc cacgccgcagcgcccggggc aggcggcggc ttccgacaag cagcggcagc 1440 aggtgcatgc atgcacgaacacctcttgcc atccatccat cgatcgccat cccgcataga 1500 atcacaagcc attgctccccaaataagtgg tgcgaggacg ccggcggcgg cgccggcggc 1560 aggagacaag aacccgcggttcctgctgca gaaggtgctc aagcagagcg acgtcggaac 1620 cctcggccgc atcgtgctccccaaagaagc ggagactcac ctgccggagc tcaagacggg 1680 ggacggcatc tcgatccccattgaggacat cggcacatct cagattttgg cccaacaaca 1740 agagcagaat gtatcttctagagaacactg gtgactttgt tcggtcgaat gagctgcagg 1800 agggtgattt catcgtgctttactctgatg tcaagtcggg caaatatctg atacgcggcg 1860 tgaaggtgag agcgcaacaggatctagcca agcacaagaa tgccagtcca gagaaaggcg 1920 gggcgtccga cgtgaaggcgggcggagaag acggcggctg caaggagaag cccccccacg 1980 gcgtccggcg atctcgccaggaggccgcct ccatgaacca gatggcggtg agcatctgaa 2040 atgagcaggc tcgccgtccgatccaccatt gaagactcag ttagctagct caagtatacc 2100 cgttgatgat gatcaaatcgatctctcgtt ctatgatccg tgcttccgtg tactgctgta 2160 gccctagtta gggatgatgatactaaagta gctatcggtc agatgtgacg ctgaagaatg 2220 catggtccgt gctgttaaacctgtataaag gctgtaaccc ttctgtacat gcatgaacat 2280 acccttattt gttgtgtgttgtcctcctaa aaaaaaaaaa aaaaaaaaaa aa 2332 3 20 DNA Artificial SequenceDescription of Artificial Sequence Primer 3 cgtcacatct gaccgatagc 20 421 DNA Artificial Sequence Description of Artificial Sequence Primer 4catctcaggt gtggagcatg c 21 5 23 DNA Artificial Sequence Description ofArtificial Sequence Primer 5 cggcacatct cagattttgg ccc 23 6 19 DNAArtificial Sequence Description of Artificial Sequence Primer 6gcggcagcag ggtgcgagg 19 7 21 DNA Artificial Sequence Description ofArtificial Sequence Primer 7 gcggcagcag gtgcatgcat g 21 8 661 PRT Avenafatua 8 Met Asp Ala Ser Ala Gly Ser Ser Pro Pro Pro His Ser Gln Glu Asn1 5 10 15 Pro Pro Lys His Gly Gly Gly Arg Gly Lys Arg Ala Gly Glu IleArg 20 25 30 Lys Gly Glu Ala Ala Thr Ala Asp Asp Phe Met Phe Ala Glu AspThr 35 40 45 Phe Pro Ser Leu Pro Asp Phe Pro Cys Leu Ser Ser Arg Ser SerSer 50 55 60 Thr Phe Ser Ser Ser Ser Ser Ser Asn Ser Ser Ser Thr His AlaAla 65 70 75 80 Ala Gly Arg Gly Val Ala Val Val Ala Asp Ala Arg Arg ArgLeu Gly 85 90 95 Glu Pro Ser Asp Pro Ala Ala Ala Gly Asp Asp Asp Val LeuAsp Asp 100 105 110 Ile Asp Glu Leu Leu Asn Ser Ala Thr Leu Ser Asp SerMet Pro Trp 115 120 125 Glu Asp Glu Pro Leu Phe Pro Asp Asp Val Gly MetMet Ile Glu Asp 130 135 140 Ala Ile Ser His Gln Pro Pro Ala Thr Gly HisArg Gly Ala Arg Asn 145 150 155 160 Ala Ala Ser Ser Glu Ala Ala Ala GlyGly Gly Gly Gln Asp Ser Ser 165 170 175 Ser Ala Asp Asp Leu Pro Arg PhePhe Met Glu Trp Leu Thr Asn Asn 180 185 190 Arg Asp Cys Ile Ser Ala GluAsp Leu Arg Ser Ile Arg Leu Arg Arg 195 200 205 Ser Thr Ile Glu Ala AlaAla Ala Arg Leu Gly Gly Gly Arg Gln Gly 210 215 220 Thr Met Gln Leu LeuLys Leu Ile Leu Thr Trp Val Gln Asn His His 225 230 235 240 Leu Gln LysLys Arg Ala Arg Val Asp Asp Glu Leu Pro Ser Pro Gly 245 250 255 Ala AsnPro Gly Tyr Glu Phe Pro Ala Glu Thr Val Ala Pro Ala Thr 260 265 270 SerTrp Leu Met Pro Tyr Gln Gln Ala Tyr Gly Arg Glu Ala Ile Tyr 275 280 285Pro Asn Ala Ala Ala Thr Gly Gln Tyr Pro Phe Gln Gln Gly Gly Ser 290 295300 Thr Ser Ser Val Val Val Ser Ser Gln Pro Phe Ser Pro Pro Ala Pro 305310 315 320 Val Ala Asp Met Gln Ala Ala Asn Met Pro Trp Pro Gln Gln TyrAla 325 330 335 Ala Phe Pro Gly Ala Ala Pro Tyr Pro Met Pro Pro Pro GlnPro Leu 340 345 350 Ala Ala Ala Gly Phe Gly Val Cys Pro Gln Pro Leu AlaGly Val Lys 355 360 365 Pro Ser Ala Ser Lys Glu Ala Arg Lys Lys Arg MetAla Arg Gln Arg 370 375 380 Arg Leu Ser Cys Leu Gln His Gln Arg Ser GlnGln Leu Asn Leu Gly 385 390 395 400 Gln Ile Gln Asn Ala Met Ile His ProGln Gln Glu Val Pro Phe Ser 405 410 415 Pro Arg Ser Ala His Ser Val ProVal Ser Pro Pro Ser Pro Gly Gly 420 425 430 Trp Cys Gly Leu Trp Pro ProPro Ser Val Gln Val Gln Gly Gln Gly 435 440 445 Gln Leu Met Val Pro AsnPro Leu Ser Thr Lys Pro Ser Ser Ser Ser 450 455 460 Arg Gln Lys Ala GlnLys Pro Ser Pro Asp Ala Gly Ala Arg Pro Pro 465 470 475 480 Ser Ser GlyAla Gln Gln Gly Ala Lys Pro Gly Ala Asp Lys Asn Leu 485 490 495 Arg PheLeu Leu Gln Lys Val Leu Lys Gln Ser Asp Val Gly Ala Leu 500 505 510 GlyArg Ile Val Leu Pro Lys Glu Ala Glu Thr His Leu Pro Glu Leu 515 520 525Lys Thr Arg Asp Gly Ile Ser Ile Pro Met Glu Asp Ile Gly Thr Ser 530 535540 Arg Val Trp Ser Met Arg Tyr Arg Phe Trp Pro Asn Asn Lys Ser Arg 545550 555 560 Met Tyr Leu Leu Glu Asn Thr Gly Asp Phe Val Arg Ser Asn GluLeu 565 570 575 Gln Glu Gly Asp Phe Ile Val Ile Tyr Ser Asp Val Lys SerGly Lys 580 585 590 Tyr Leu Ile Arg Gly Val Lys Val Arg Pro Pro Gln AspLeu Ala Lys 595 600 605 Gln Lys His Gly Ser Leu Glu Lys Gly Ser Thr SerAsp Ala Met Pro 610 615 620 Cys Ala Glu Asp Gly Gly Ala Glu Ala Gly GlyCys Lys Gly Lys Ser 625 630 635 640 Pro His Gly Val Arg Arg Ser Arg GlnGlu Ala Ala Ser Met Asn Gln 645 650 655 Met Ala Val Ser Ile 660 9 691PRT Zea mays 9 Met Glu Ala Ser Ser Gly Ser Ser Pro Pro His Ser Gln GluAsn Pro 1 5 10 15 Pro Glu His Gly Gly Asp Met Gly Gly Ala Pro Ala GluGlu Ile Gly 20 25 30 Gly Glu Ala Ala Asp Asp Phe Met Phe Ala Glu Asp ThrPhe Pro Ser 35 40 45 Leu Pro Asp Phe Pro Cys Leu Ser Ser Pro Ser Ser SerThr Phe Ser 50 55 60 Ser Asn Ser Ser Ser Asn Ser Ser Ser Ala Tyr Thr AsnThr Ala Gly 65 70 75 80 Arg Ala Gly Gly Glu Pro Ser Glu Pro Ala Ser AlaGly Glu Gly Phe 85 90 95 Asp Ala Leu Asp Asp Ile Asp Gln Leu Leu Asp PheAla Ser Leu Ser 100 105 110 Met Pro Trp Asp Ser Glu Pro Phe Pro Gly ValSer Met Met Leu Glu 115 120 125 Asn Ala Met Ser Ala Pro Pro Gln Pro ValGly Asp Gly Met Ser Glu 130 135 140 Glu Lys Ala Val Pro Glu Gly Thr ThrGly Gly Glu Glu Ala Cys Met 145 150 155 160 Asp Ala Ser Glu Gly Glu GluLeu Pro Arg Phe Phe Met Glu Trp Leu 165 170 175 Thr Ser Asn Arg Glu AsnIle Ser Ala Glu Asp Leu Arg Gly Ile Arg 180 185 190 Leu Arg Arg Ser ThrIle Glu Ala Ala Ala Ala Arg Leu Gly Gly Gly 195 200 205 Arg Gln Gly ThrMet Gln Leu Leu Lys Leu Ile Leu Thr Trp Val Gln 210 215 220 Asn His HisLeu Gln Arg Lys Arg Pro Arg Asp Val Met Glu Glu Glu 225 230 235 240 AlaGly Leu His Val Gln Leu Pro Ser Pro Val Ala Asn Pro Pro Gly 245 250 255Tyr Glu Phe Pro Ala Gly Gly Gln Asp Met Ala Ala Gly Gly Gly Thr 260 265270 Ser Trp Met Pro His Gln Gln Ala Phe Thr Pro Pro Ala Ala Tyr Gly 275280 285 Gly Asp Ala Val Tyr Pro Ser Ala Ala Gly Gln Gln Tyr Ser Phe His290 295 300 Gln Gly Pro Ser Thr Ser Ser Val Val Val Asn Ser Gln Pro PheSer 305 310 315 320 Pro Pro Pro Val Gly Asp Met His Gly Ala Asn Met AlaTrp Pro Gln 325 330 335 Gln Tyr Val Pro Phe Pro Pro Pro Gly Ala Ser ThrGly Ser Tyr Pro 340 345 350 Met Pro Gln Pro Phe Ser Pro Gly Phe Gly GlyGln Tyr Ala Gly Ala 355 360 365 Gly Ala Gly His Leu Ser Val Ala Pro GlnArg Met Ala Gly Val Glu 370 375 380 Ala Ser Ala Thr Lys Glu Ala Arg LysLys Arg Met Ala Arg Gln Arg 385 390 395 400 Arg Leu Ser Cys Leu Gln GlnGln Arg Ser Gln Gln Leu Ser Leu Gly 405 410 415 Gln Ile Gln Thr Ser ValHis Leu Gln Glu Pro Ser Pro Arg Ser Thr 420 425 430 His Ser Gly Pro ValThr Pro Ser Ala Gly Gly Trp Gly Phe Trp Ser 435 440 445 Pro Ser Ser GlnGln Gln Val Gln Asn Pro Leu Ser Lys Ser Asn Ser 450 455 460 Ser Arg AlaPro Pro Ser Ser Leu Glu Ala Ala Ala Ala Ala Pro Gln 465 470 475 480 ThrLys Pro Ala Pro Ala Gly Ala Arg Gln Asp Asp Ile His His Arg 485 490 495Leu Ala Ala Ala Ser Asp Lys Arg Gln Gly Ala Lys Ala Asp Lys Asn 500 505510 Leu Arg Phe Leu Leu Gln Lys Val Leu Lys Gln Ser Asp Val Gly Ser 515520 525 Leu Gly Arg Ile Val Leu Pro Lys Lys Glu Ala Glu Val His Leu Pro530 535 540 Glu Leu Lys Thr Arg Asp Gly Ile Ser Ile Pro Met Glu Asp IleGly 545 550 555 560 Thr Ser Arg Val Trp Asn Met Arg Tyr Arg Phe Trp ProAsn Asn Lys 565 570 575 Ser Arg Met Tyr Leu Leu Glu Asn Thr Gly Glu PheVal Arg Ser Asn 580 585 590 Glu Leu Gln Glu Gly Asp Phe Ile Val Ile TyrSer Asp Val Lys Ser 595 600 605 Gly Lys Tyr Leu Ile Arg Gly Val Lys ValArg Pro Pro Pro Ala Gln 610 615 620 Glu Gln Gly Ser Gly Ser Ser Gly GlyGly Lys His Arg Pro Leu Cys 625 630 635 640 Pro Ala Gly Pro Glu Arg AlaAla Ala Ala Gly Ala Pro Glu Asp Ala 645 650 655 Val Val Asp Gly Val SerGly Ala Cys Lys Gly Arg Ser Pro Glu Gly 660 665 670 Val Arg Arg Val ArgGln Gln Gly Ala Gly Ala Met Ser Gln Met Ala 675 680 685 Val Ser Ile 69010 728 PRT Oryza sativa 10 Met Asp Ala Ser Ala Gly Ser Ser Ala Pro HisSer His Gly Asn Pro 1 5 10 15 Gly Lys Gln Gly Gly Gly Gly Gly Gly GlyGly Gly Arg Gly Lys Ala 20 25 30 Pro Ala Ala Glu Ile Arg Gly Glu Ala AlaArg Asp Asp Val Phe Phe 35 40 45 Ala Asp Asp Thr Phe Pro Leu Leu Pro AspPhe Pro Cys Leu Ser Ser 50 55 60 Pro Ser Ser Ser Thr Phe Ser Ser Ser SerSer Ser Asn Ser Ser Ser 65 70 75 80 Ala Phe Thr Thr Ala Ala Gly Gly GlyCys Gly Gly Glu Pro Ser Glu 85 90 95 Pro Ala Ser Ala Ala Asp Gly Phe GlyGlu Leu Ala Asp Ile Asp Gln 100 105 110 Leu Leu Asp Leu Ala Ser Leu SerVal Pro Trp Glu Ala Glu Gln Pro 115 120 125 Leu Phe Pro Asp Asp Val GlyMet Met Ile Glu Asp Ala Met Ser Gly 130 135 140 Gln Pro His Gln Ala AspAsp Cys Thr Gly Asp Gly Asp Thr Lys Ala 145 150 155 160 Val Met Glu AlaAla Gly Gly Gly Asp Asp Ala Gly Asp Ala Cys Met 165 170 175 Glu Gly SerAsp Ala Pro Asp Asp Leu Pro Ala Phe Phe Met Glu Trp 180 185 190 Leu ThrSer Asn Arg Glu Tyr Ile Ser Ala Asp Asp Leu Arg Ser Ile 195 200 205 ArgLeu Arg Arg Ser Thr Ile Glu Ala Ala Ala Ala Arg Leu Gly Gly 210 215 220Gly Arg Gln Gly Thr Met Gln Leu Leu Lys Leu Ile Leu Thr Trp Val 225 230235 240 Gln Asn His His Leu Gln Lys Lys Arg Pro Arg Thr Ala Ile Asp Asp245 250 255 Gly Ala Ala Ser Ser Asp Pro Gln Leu Pro Ser Pro Gly Ala AsnPro 260 265 270 Gly Tyr Glu Phe Pro Ser Gly Gly Gln Glu Met Gly Ser AlaAla Ala 275 280 285 Thr Ser Trp Met Pro Tyr Gln Ala Phe Thr Pro Pro AlaAla Tyr Gly 290 295 300 Gly Asp Ala Met Tyr Pro Gly Ala Ala Gly Pro PhePro Phe Gln Gln 305 310 315 320 Ser Cys Ser Lys Ser Ser Val Val Val SerSer Gln Pro Phe Ser Pro 325 330 335 Pro Thr Ala Ala Ala Ala Gly Asp MetHis Ala Ser Gly Gly Gly Asn 340 345 350 Met Ala Trp Pro Gln Gln Phe AlaPro Phe Pro Val Ser Ser Thr Ser 355 360 365 Ser Tyr Thr Met Pro Ser ValVal Pro Pro Pro Phe Thr Ala Gly Phe 370 375 380 Pro Gly Gln Tyr Ser GlyGly His Ala Met Cys Ser Pro Arg Leu Ala 385 390 395 400 Gly Val Glu ProSer Ser Thr Lys Glu Ala Arg Lys Lys Arg Met Ala 405 410 415 Arg Gln ArgArg Leu Ser Cys Leu Gln Gln Gln Arg Ser Gln Gln Leu 420 425 430 Asn LeuSer Gln Ile His Ile Ser Gly His Pro Gln Glu Pro Ser Pro 435 440 445 ArgAla Ala His Ser Ala Pro Val Thr Pro Ser Ser Ala Gly Cys Arg 450 455 460Ser Trp Gly Ile Trp Pro Pro Ala Ala Gln Ile Ile Gln Asn Pro Leu 465 470475 480 Ser Asn Lys Pro Asn Pro Pro Pro Ala Thr Ser Lys Gln Pro Lys Pro485 490 495 Ser Pro Glu Lys Pro Lys Pro Lys Pro Gln Ala Ala Ala Thr AlaGly 500 505 510 Ala Glu Ser Leu Gln Arg Ser Thr Ala Ser Glu Lys Arg GlnAla Lys 515 520 525 Thr Asp Lys Asn Leu Arg Phe Leu Leu Gln Lys Val LeuLys Gln Ser 530 535 540 Asp Val Gly Ser Leu Gly Arg Ile Val Leu Pro LysLys Glu Ala Glu 545 550 555 560 Val His Leu Pro Glu Leu Lys Thr Arg AspGly Val Ser Ile Pro Met 565 570 575 Glu Asp Ile Gly Thr Ser Gln Val TrpAsn Met Arg Tyr Arg Phe Trp 580 585 590 Pro Asn Asn Lys Ser Arg Met TyrLeu Leu Glu Asn Thr Gly Asp Phe 595 600 605 Val Arg Ser Asn Glu Leu GlnGlu Gly Asp Phe Ile Val Ile Tyr Ser 610 615 620 Asp Ile Lys Ser Gly LysTyr Leu Ile Arg Gly Val Lys Val Arg Arg 625 630 635 640 Ala Ala Gln GluGln Gly Asn Ser Ser Gly Ala Val Gly Lys His Lys 645 650 655 His Gly SerPro Glu Lys Pro Gly Val Ser Ser Asn Thr Lys Ala Ala 660 665 670 Gly AlaGlu Asp Gly Thr Gly Gly Asp Asp Ser Ala Glu Ala Ala Ala 675 680 685 AlaAla Ala Ala Gly Lys Ala Asp Gly Gly Gly Cys Lys Gly Lys Ser 690 695 700Pro His Gly Val Arg Arg Ser Arg Gln Glu Ala Ala Ala Ala Ala Ser 705 710715 720 Met Ser Gln Met Ala Val Ser Ile 725 11 720 PRT Arabidopsisthaliana 11 Met Lys Ser Leu His Val Ala Ala Asn Ala Gly Asp Leu Ala GluAsp 1 5 10 15 Cys Gly Ile Leu Gly Gly Asp Ala Asp Asp Thr Val Leu MetAsp Gly 20 25 30 Ile Asp Glu Val Gly Arg Glu Ile Trp Leu Asp Asp His GlyGly Asp 35 40 45 Asn Asn His Val His Gly His Gln Asp Asp Asp Leu Ile ValHis His 50 55 60 Asp Pro Ser Ile Phe Tyr Gly Asp Leu Pro Thr Leu Pro AspPhe Pro 65 70 75 80 Cys Met Ser Ser Ser Ser Ser Ser Ser Thr Ser Pro AlaPro Val Asn 85 90 95 Ala Ile Val Ser Ser Ala Ser Ser Ser Ser Ala Ala SerSer Ser Thr 100 105 110 Ser Ser Ala Ala Ser Trp Ala Ile Leu Arg Ser AspGly Glu Asp Pro 115 120 125 Thr Pro Asn Gln Asn Gln Tyr Ala Ser Gly AsnCys Asp Asp Ser Ser 130 135 140 Gly Ala Leu Gln Ser Thr Ala Ser Met GluIle Pro Leu Asp Ser Ser 145 150 155 160 Gln Gly Phe Gly Cys Gly Glu GlyGly Gly Asp Cys Ile Asp Met Met 165 170 175 Glu Thr Phe Gly Tyr Met AspLeu Leu Asp Ser Asn Glu Phe Phe Asp 180 185 190 Thr Ser Ala Ile Phe SerGln Asp Asp Asp Thr Gln Asn Pro Asn Leu 195 200 205 Met Asp Gln Thr LeuGlu Arg Gln Glu Asp Gln Val Val Val Pro Met 210 215 220 Met Glu Asn AsnSer Gly Gly Asp Met Gln Met Met Asn Ser Ser Leu 225 230 235 240 Glu GlnAsp Asp Asp Leu Ala Ala Val Phe Leu Glu Trp Leu Lys Asn 245 250 255 AsnLys Glu Thr Val Ser Ala Glu Asp Leu Arg Lys Val Lys Ile Lys 260 265 270Lys Ala Thr Ile Glu Ser Ala Ala Arg Arg Leu Gly Gly Gly Lys Glu 275 280285 Ala Met Lys Gln Leu Leu Lys Leu Ile Leu Glu Trp Val Gln Thr Asn 290295 300 His Leu Gln Arg Arg Arg Thr Thr Thr Thr Thr Thr Asn Leu Ser Tyr305 310 315 320 Gln Gln Ser Phe Gln Gln Asp Pro Phe Gln Asn Pro Asn ProAsn Asn 325 330 335 Asn Asn Leu Ile Pro Pro Ser Asp Gln Thr Cys Phe SerPro Ser Thr 340 345 350 Trp Val Pro Pro Pro Pro Gln Gln Gln Ala Phe ValSer Asp Pro Gly 355 360 365 Phe Gly Tyr Met Pro Ala Pro Asn Tyr Pro ProGln Pro Glu Phe Leu 370 375 380 Pro Leu Leu Glu Ser Pro Pro Ser Trp ProPro Pro Pro Gln Ser Gly 385 390 395 400 Pro Met Pro His Gln Gln Phe ProMet Pro Pro Thr Ser Gln Tyr Asn 405 410 415 Gln Phe Gly Asp Pro Thr GlyPhe Asn Gly Tyr Asn Met Asn Pro Tyr 420 425 430 Gln Tyr Pro Tyr Val ProAla Gly Gln Met Arg Asp Gln Arg Leu Leu 435 440 445 Arg Leu Cys Ser SerAla Thr Lys Glu Ala Arg Lys Lys Arg Met Ala 450 455 460 Arg Gln Arg ArgPhe Leu Ser His His His Arg His Asn Asn Asn Asn 465 470 475 480 Asn AsnAsn Asn Asn Asn Gln Gln Asn Gln Thr Gln Ile Gly Glu Thr 485 490 495 CysAla Ala Val Ala Pro Gln Leu Asn Pro Val Ala Thr Thr Ala Thr 500 505 510Gly Gly Thr Trp Met Tyr Trp Pro Asn Val Pro Ala Val Pro Pro Gln 515 520525 Leu Pro Pro Val Met Glu Thr Gln Leu Pro Thr Met Asp Arg Ala Gly 530535 540 Ser Ala Ser Ala Met Pro Arg Gln Gln Val Val Pro Asp Arg Arg Gln545 550 555 560 Gly Trp Lys Pro Glu Lys Asn Leu Arg Phe Leu Leu Gln LysVal Leu 565 570 575 Lys Gln Ser Asp Val Gly Asn Leu Gly Arg Ile Val LeuPro Lys Lys 580 585 590 Glu Ala Glu Thr His Leu Pro Glu Leu Glu Ala ArgAsp Gly Ile Ser 595 600 605 Leu Ala Met Glu Asp Ile Gly Thr Ser Arg ValTrp Asn Met Arg Tyr 610 615 620 Arg Phe Trp Pro Asn Asn Lys Ser Arg MetTyr Leu Leu Glu Asn Thr 625 630 635 640 Gly Asp Phe Val Lys Thr Asn GlyLeu Gln Glu Gly Asp Phe Ile Val 645 650 655 Ile Tyr Ser Asp Val Lys CysGly Lys Tyr Leu Ile Arg Gly Val Lys 660 665 670 Val Arg Gln Pro Ser GlyGln Lys Pro Glu Ala Pro Pro Ser Ser Ala 675 680 685 Ala Thr Lys Arg GlnAsn Lys Ser Gln Arg Asn Ile Asn Asn Asn Ser 690 695 700 Pro Ser Ala AsnVal Val Val Ala Ser Pro Thr Ser Gln Thr Val Lys 705 710 715 720 12 750PRT Phaseolus vulgaris 12 Met Glu Cys Glu Val Lys Leu Lys Gly Gly AspLeu His Ala Glu Gly 1 5 10 15 Val Thr Glu Thr Asn Ala Val Gly Phe AspAla Met Glu Asp Glu Gln 20 25 30 Thr Leu Thr Val Ala Glu Arg Glu Met TrpLeu Asn Ser Asp Gln Asp 35 40 45 Glu Phe Leu Gly Val Asn Glu Ala Ser MetPhe Tyr Ala Asn Phe Pro 50 55 60 Pro Leu Pro Asp Phe Pro Cys Thr Ser SerSer Ser Ser Ser Ser Ser 65 70 75 80 Ala Ala Pro Leu Pro Leu Lys Thr ThrThr Cys Ser Thr Thr Thr Thr 85 90 95 Ala Thr Thr Ala Thr Ser Ser Ser SerSer Ser Ser Ser Trp Ala Val 100 105 110 Leu Lys Ser Asp Val Glu Glu GluAsp Val Glu Lys Asn His Cys Asn 115 120 125 Gly Ser Met Gln Asp Gln PheAsp Ala Thr Ala Leu Ser Ser Thr Ala 130 135 140 Ser Met Glu Ile Ser GlnGln Gln Asn Pro Asp Pro Gly Leu Gly Gly 145 150 155 160 Ser Val Gly GluCys Met Glu Asp Val Met Asp Thr Phe Gly Tyr Met 165 170 175 Glu Leu LeuGlu Ala Asn Asp Phe Phe Asp Pro Ala Ser Ile Phe Gln 180 185 190 Asn GluGlu Ser Glu Asp Pro Leu Ile Glu Phe Gly Val Leu Glu Glu 195 200 205 GlnVal Ser Leu Gln Glu Glu Gln His Glu Met Val His Gln Gln Glu 210 215 220Asn Thr Glu Glu Asp Arg Lys Val Pro Val Cys Glu Val Ile Lys Gly 225 230235 240 Glu Glu Glu Gly Gly Gly Gly Gly Gly Gly Arg Val Val Asp Asp Glu245 250 255 Met Ser Asn Val Phe Leu Glu Trp Ser Lys Ser Asn Lys Asp SerVal 260 265 270 Ser Ala Asn Asp Leu Arg Asn Val Lys Leu Lys Lys Ala ThrIle Glu 275 280 285 Ser Ala Ala Lys Arg Leu Gly Gly Gly Lys Glu Ala MetLys Gln Leu 290 295 300 Leu Lys Leu Ile Leu Glu Trp Val Gln Thr Ser HisLeu Gln Asn Lys 305 310 315 320 Arg Arg Lys Glu Asn Gly Ser Asn Ala LeuGln Ala Thr Phe Gln Asp 325 330 335 Pro Ser Ala Gln Thr Lys Glu Asn AlaHis Thr Ser Gly Ser Phe Ala 340 345 350 Pro Glu Ser Asn Ser Cys Phe AsnAsn Gln Thr Pro Trp Leu Asn Pro 355 360 365 Gln Thr Phe Gly Thr Asp GlnAla Pro Val Met Val Pro Ser Gln Pro 370 375 380 Tyr Ser Gln Pro Val AlaGly Tyr Val Gly Asp Pro Tyr Thr Ser Gly 385 390 395 400 Ser Ala Pro AsnAsn Ile Thr Val Asn His Asn His Asn Asn Asn Pro 405 410 415 Tyr Gln ProGly Thr Asp Gln Tyr His Met Leu Glu Ser Ala His Ser 420 425 430 Trp ProHis Ser Gln Phe Asn Val Ala Ser His Tyr Ser Gln Ser Tyr 435 440 445 GlyGlu Asn Gly Leu Phe Thr His Gly Gly Phe Gly Gly Tyr Ala Ile 450 455 460Thr Arg Tyr Pro Tyr Gln Phe Phe His Gly Pro Gly Asp Arg Leu Met 465 470475 480 Arg Leu Gly Pro Ser Ala Thr Lys Glu Ala Arg Lys Lys Arg Met Ala485 490 495 Arg Gln Arg Lys Phe Leu Ser His His Arg Asn Gln Asn Gly AsnHis 500 505 510 Leu Gln Asn Gln Gly Ser Asp Pro His Ala Arg Leu Gly AsnAsp Asn 515 520 525 Cys Thr Thr Gly Leu Val Ala Pro His Gln Pro Asn SerAla Ala Ala 530 535 540 Asn Trp Met Tyr Trp Gln Ala Met Thr Gly Gly ProAla Gly Pro Leu 545 550 555 560 Ala Pro Val Val Pro Ala Asp Pro Leu AlaGly Gln Thr Val Val Asp 565 570 575 Arg Thr Thr Met His Thr Gln Asn SerHis Gln Asn Arg Ala Ala Ser 580 585 590 Asp Arg Arg Gln Gly Trp Lys ProGlu Lys Asn Val Arg Phe Leu Gly 595 600 605 Gln Lys Val Leu Lys Gln SerAsp Val Gly Lys Leu Gly Arg Ile Val 610 615 620 Leu Pro Lys Lys Glu AlaGlu Thr His Leu Pro Glu Leu Glu Ala Arg 625 630 635 640 Asp Gly Ile SerIle Thr Met Glu Asp Ile Gly Thr Ser Arg Val Trp 645 650 655 Asn Met ArgTyr Arg Tyr Trp Pro Asn Asn Lys Ser Arg Met Tyr Met 660 665 670 Leu GluAsn Thr Gly Asp Phe Val Arg Ala Asn Gly Leu Gln Glu Gly 675 680 685 AspPhe Ile Val Ile Tyr Ser Asp Val Lys Cys Gly Lys Tyr Met Ile 690 695 700Arg Gly Val Lys Val Arg Gln Gln Gly Val Lys Pro Glu Thr Lys Pro 705 710715 720 Ala Gly Lys Ser Gln Lys Thr Thr Thr Gly Thr Asn Ala Ser Tyr Thr725 730 735 Ala Gly Thr Ala Ala Asn Asn Gly Met Ser Ser His Arg Asn 740745 750 13 817 DNA Triticum aestivum 13 attctcggga cccccgagcc agaggcgcctcggcgggccc cacgccgcag cgcccggggc 60 aggcggcggc ttccgacaag cagcggcagcagggtgcgag gacccggcgg cggcgccggc 120 ggcaggagac aagaacccgc ggttcctgctgcagaaggtg ctcaagcaga gcgacgtcgg 180 aacctcggcc gcatcgtgct ccccaaaaaggaagcggaga ctcacctgcc ggagctcaag 240 acgggggacg gcatctcgat ccccattgaggacatcggca catctcagat tttggcccaa 300 caacaagagc agaatgtatc ttctagagaacactggtgac tttgttcggt cgaatagctg 360 caggagggtg atttcatcgt gctttactctgatgtcaagt cggcaaatat ctatccggcg 420 tgaaggtgag agcgcaacag gatctagccaagcacaaaat gccagtccag agaaaggcgg 480 ggcttcctga agcgggcgga gaagacggcggctgcaggag aagccccccc acggcgtccg 540 gcgatctcgc caggaggccg cctccatgaaccagatggcg gtgagcatct gaaatgagca 600 gctcgccgtc cgatccacca ttgaagatcagttagctagc tcaagtatac ccttgatgat 660 gatcaaatcg atctctcgtt tagatccgtgcttcggtatg ctgtagccct agttagggat 720 gatgatacta aagtactatc ggtcagatgtgacctaaaat gcatggtccg tgctgttaac 780 cgtataagct gtaacccttt taaaaaaaaaaaaaaaa 817 14 1133 DNA Triticum aestivum 14 ggcacgaggc ggcgcctgtcgtgcctgcag cagcagcgga gccagcagct gaatctgagc 60 cagatccaaa ccggcggcttccctcaagag ccatcccccc gcgcggcgca ctcggcgccg 120 gtcactccgc cgtcgtctggctggggaggc ctctggacgc aacaagccgt ccagagccag 180 ccccatggcc agctcatggtccaggtcccg aatccgctgt cgacgaagtc caattcctca 240 aggcagaagc agcaaaaaccctcgccggac gcagcagcga ggccgccctc cggcggcgcc 300 gccacgccgc agcgcccggggcaggcggcg gcttccgaca agcagcggca gcagggtgcg 360 aggacgccgg cggcggcgccggcggcagga gacaagaacc cgcggttcct gctgcagaag 420 gtgctcaagc agagcgacgtcggaaccctc ggccgcatcg tgctccccaa agaagcggag 480 actcacctgc cggagctcaagacgggggac ggcatctcga tccccattga ggacatcggc 540 acatctcaga ttttggcccaacaacaagag cagaatgtat cttctagaga acactggtga 600 ctttgttcgg tcgaatgagctgcaggaggg tgatttcatc gtgctttact ctgatgtcaa 660 gtcgggcaaa tatctgatacgcggcgtgaa ggtgagagcg caacaggatc tagccaagca 720 caagaatgcc agtccagagaaaggcggggc gtccgacgtg aaggcgggcg gagaagacgg 780 cggctgcaag gagaagcccccccactgcgt ccggcgatct cgccaggagg ccgcctccat 840 gaaccagatg gcggtgagcatctgaaatga gcaggctcgc cgtccgatcc acattgaaga 900 ctcagttagc tagctcaagtatacccgttg atgatgatca aatcgatctc tcgttctatg 960 atccgtgctt ccgtgtactgctgtagccct agttagggat gatgatacta aagtagctat 1020 cggtcagatg tgacgctgaagaatgcatgg tccgtgctgt taaacctgta taaaggctgt 1080 aacccttctg tacatgcatgaacataccct taaaaaaaaa aaaaaaaaaa aaa 1133 15 1185 DNA Triticum aestivum15 cgcagcggat ggaaccggcg gcgaccaggg aggcccggaa gaagaggatg gcgaggcagc 60ggcgcctgtc gtgcctgcag cagcagcgga gccagcagct gaatctgagc cagatccaaa 120gcggcggctt ccctcaagaa ccatcccccc gcgcggcgca ctcggcgccg gtcacgccgc 180cctcttccgg ctggggaggc ctctggtcgc agcatgccgt ccagggccag ccccatggcc 240agctcatggt ccaggttccg aatccgctgt cgacgaagtc caattcctcg aggcagaagc 300agcaaaaacc ctcgccggat gcagcagcga ggccgccctc cggcggcgcc gccacgcagc 360agcgcccggg gcaggcggcg gcttccgaca agcagcggca gcagggtgcg aggacgccgg 420cggcggcgcc ggcggcagga gacaagaacc tgcggttcct gctgcagaag gtgctcaagc 480agagcgacgt cggaaccctc ggccgcatcg tgctccccaa agaagcggag actcacctgc 540cggagctcaa gacgggggac ggcatctcga tccccattga ggacatcggc acatctcagg 600tgtggagcat gcggtaccga ttttggccca acaacaagag cagaatgtat ctttctggag 660aacactggag actttgttcg gtcgaatgag ctgcaggagg gtgatttcat cgtgctttac 720tctgatgtca agtcgggcaa atatctgata cgcggcgtga aggtaagagc gcaacaggat 780ctagccaagc acaagaatgg cagtccagag aaaggtgggg cgtccgacgc gaaggcgggc 840gcagaagacg gtggttgcaa agagaagtct ccgcacggtg tccggcgatc tcgccaggag 900gccgcctcca tgaaccagat ggccgtgagc atctgaaatg agcaggctcg cgcggtccga 960tcccccattg aagactactt agctagctca agtatacctg ttgatgatga tcaaatcgat 1020ctcccgttct atgatccgtg cttccgtgta ctgctgtagc cctagttagg gatggtgata 1080ctaaagtagc tatcggtcag atgtgacgct gaagaatgca tggtccgtgc tgttaaacct 1140gtataaaagt gtaaccttct gttaaaaaaa aaaaaaaaaa aaaaa 1185 16 1916 DNATriticum aestivum 16 ggcacgagcc accatcgagg ccgcggccgc gcgcctcggtggggggcgcc agggcaccat 60 gcagctgctc aagctcatcc tcacctgggt gcagaaccaccacctgcaga agaagcgccc 120 ccgcgtcggc gccatggatc aggaggcgcc gccggcaggaggccagctcc ccagccccgg 180 cgcaaacccc ggctacgaat tccccgcgga gacgggtgccgccgctaaca catcttggat 240 gccctaccag gccttctcgc caactggatc ctacggcggcgaggcgatct acccgttcca 300 gcagggctgc agcacgagca gcgtggccgt gagcagccagccgttctccc cgccggcggc 360 gcccgacatg cacgccgggg cctggccgct tcagtacgcggcgttcgtcc cagctggggc 420 cacatccgca ggcactcaaa catacccgat gccgccgccgggggccgtgc cgcagccgtt 480 cgcggccccc ggattcgccg ggcagttccc gcagcggatggaaccggcgg cgaccaggga 540 ggcccggaag aagaggatgg cgaggcagcg gcgcctgtcgtgcctgcagc agcagcggag 600 ccagcagctg aatctgagcc agatccaaag cggcggcttccctcaagaac catccccccg 660 cgcggcgcac tcggcgccgg tcacgccgcc ctcttccggctggggaggcc tctggtcgca 720 gcatgccgtc cagggccagc cccatggcca gctcatggtccaggttccga atccgctgtc 780 gacgaagtcc aattcctcga ggcagaagca gcaaaaaccctcgccggatg cagcagcgag 840 gccgccctcc ggccggccgc cacgcagcag cgcccggggcaggcggcggc ttccgacaag 900 cagcggcagc aggtgcatgc atgcacgaac acctcttgccatccatccat cgatcgccat 960 cccgcataga atcacaagcc attgctcccc aaataagtgtgcgtacatcg taagagacgc 1020 acatcgctgt ccagcgatag gatatccccg catcgccatcccgcatagaa tcacaagcca 1080 ttgctcccct gcacggtgaa ttgcgtttct caacgaggttccgtgcatgc gcgcagggtg 1140 cgaggacgcc ggcggcggcg ccggcggcag gagacaagaacctgcggttc ctgctgcaga 1200 aggtgctcaa gcagagcgac gtcggaaccc tcggccgcatcgtgctcccc aaaaaggaag 1260 cggagactca cctgccggag ctcaagacgg gggacggcatctcgatcccc attgaggaca 1320 tcggcacatc tcaggtgtgg agcatgcggt accgattttggcccaacaac aagagcagaa 1380 tgtatgttgt ggagaacact ggagactttg ttcggtcgaatgagctgcag gagggtgatt 1440 tcatcgtgct ttactctgat gtcaagtcgg gcaaatatctgatacgcggc gtgaaggtaa 1500 gagcgcaaca ggatctagcc aagcacaaga atggcagtccagagaaaggt ggggcgtccg 1560 acgcgaaggc gggcgcagaa gacggtggtt gcaaagagaagtctccgcac ggtgtccggc 1620 gatctcgcca ggaggccgcc tccatgaacc agatggccgtgagcatctga aatgagcagg 1680 ctcgcgcggt ccgatccccc attgaagact acttagctagctcaagtata cctgttgatg 1740 atgatcaaat cgatctcccg ttctatgatc cgtgcttccgtgtactgctg tagccctagt 1800 tagggatggt gatactaaag tagctatcgg tcagatgtgacgctgaagaa tgcatggtcc 1860 gtgctgttaa acctgtataa aggctgtaac ccttctgtaaaaaaaaaaaa aaaaaa 1916 17 953 DNA Triticum aestivum 17 gtccagagccagccccatgg ccagctcatg gtccaggtcc cgaatccgct gtcgacgaag 60 tccaattcctcaaggcagaa gcagcaaaaa ccctcgccgg acgcagcagc gaggccgccc 120 tccggcggcgccgccacgcc gcagcgcccg gggcaggcgg cggcttccga caagcagcgg 180 cagcagggtgcgaggacgcc ggcggcggcg ccggcggcag gagacaagaa cccgcggttc 240 ctgctgcagaaggtgctcaa gcagagcgac gtcggaaccc tcggccgcat cgtgctcccc 300 aaagaagcggagactcacct gccggagctc aagacggggg acggcatctc gatccccatt 360 gaggacatcggcacatctca ggtgtggagc atgcgatttt ggcccaacaa caagagcaga 420 atgtatcttctagagaacac tggtgacttt gttcggtcga atgagctgca ggagggtgat 480 ttcatcgtgctttactctga tgtcaagtcg ggcaaatatc tgatacgcgg cgtgaaggtg 540 agagcgcaacaggatctagc caagcacaag aatgccagtc cagagaaagg cggggcgtcc 600 gacgtgaaggcgggcggaga agacggcggc tgcaaggaga agccccccca cggcgtccgg 660 cgatctcgccaggaggccgc ctccatgaac cagatggcgg tgagcatctg aaatgagcag 720 gctcgccgtccgatccacca ttgaagactc agttagctag ctcaagtata cccgttgatg 780 atgatcaaatcgatctctcg ttctatgatc cgtgcttccg tgtactgctg tagccctagt 840 tagggatgatgatactaaag tagctatcgg tcagatgtga cgctgaagaa tgcatggtcc 900 gtgctgttaaacctgtaaaa gaaaaaaaaa aaaaaagaaa aagaaaaaaa aaa 953 18 1001 DNA Triticumaestivum 18 tggtcgcagc atgccgtcca gggccagccc catggccagc tcatggtccaggttccgaat 60 ccgctgtcga cgaagtccaa ttcctcgagg cagaagcagc aaaaaccctcgccggatgca 120 gcagcgaggc cgccctccgg cggcgccgcc acgcagcagc gcccggggcaggcggcggct 180 tccgacaagc agcggcagca gggtgcgagg acgccggcgg cggcgccggcggcaggagac 240 aagaacctgc ggttcctgct gcagaaggtg ctcaagcaga gcgacgtcggaaccctcggc 300 cgcatcgtgc tccccaaaaa ggaagcggag actcacctgc cggagctcaagacgggggac 360 ggcatctcga tccccattga ggacatcggc acatctcagg tgtggagcatgcggtaccga 420 ttttggccca acaacaagag cagaatgtat cttctggaga acactggagactttgttcgg 480 tcgaatgagc tgcaggaggg tgatttcatc gtgctttact ctgatgtcaagtcgggcaaa 540 tatctgatac gcggcgtgaa ggtaagagcg caacaggatc tagccaagcacaagaatggc 600 agtccagaga aaggtggggc gtccgacgcg aaggcgggcg cagaagacggtggttgcaaa 660 gagaagtctc cgcacggtgt ccggcgatct cgccaggagg ccgcctccatgaaccagatg 720 gccgtgagca tctgaaatga gcaggctcgc gcggtccgat cccccattgaagactactta 780 gctagctcaa gtatacctgt tgatgatgat caaatcgatc tcccgttctatgatccgtgc 840 ttccgtgtac tgctgtagcc ctagttaggg atggtgatac taaagtagctatcggtcaga 900 tgtgacgctg aagaatgcat ggtccgtgct gttaaacctg tataaaggctgtaacccttc 960 tgtaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa a 1001 191173 DNA Triticum aestivum 19 ggcacgagcc gcagcggatg gagccggcggcgaccaggga ggcccggaag aagaggatgg 60 cgaggcagcg gcgcctgtcg tgcctgcagcagcagcggag ccagcagctg aatctgagcc 120 agatccaaac cggcggcttc cctcaagagccatccccccg cgcggcgcac tcggcgccgg 180 tcacgccgcc gtcgtctggc tggggaggcctctggacgca acaagccgtc cagagccagc 240 cccatggcca gctcatggtc caggtcccgaatccgctgtc gacgaagtcc aattcctcaa 300 ggcagaagca gcaaaaaccc tcgccggacgcagcagcgag gccgccctcc ggcggcgccg 360 ccacgccgca gcgcccgggg caggcggcggcttccgacaa gcagcggcag cagggtgcga 420 ggacgccggc ggcggcgccg gcggcaggagacaagaaccc gcggttcctg ctgcagaagg 480 tgctcaagca gagcgacgtc ggaaccctcggccgcatcgt gctccccaaa aaggaagcgg 540 agactcacct gccggagctc aagacgggggacggcatctc gatccccatt gaggacatcg 600 gcacatctca gattttggcc caacaacaagagcagaatgt atcttctaga gaacactggt 660 gactttgttc ggtcgaatga gctgcaggagggtgatttca tcgtgcttta ctctgatgtc 720 aagtcgggca aatatctgat acgcggcgtgaaggtgagag cgcaacagga tctagccaag 780 cacaagaatg ccagtccaga gaaaggcggggcgtccgacg tgaaggcggg cggagaagac 840 ggcggctgca aggagaagcc cccccacggcgtccggcgat ctcgccagga ggccgcctcc 900 atgaaccaga tggcggtgag catctgaaatgagcaggctc gccgtccgat ccaccattga 960 agactcagtt agctagctca agtatacccgttgatgatga tcaaatcgat ctctcgttct 1020 atgatccgtg cttccgtgta ctgctgtagccctagttagg gatgatgata ctaaagtagc 1080 tatcggtcag atgtgacgct gaagaatgcatggtccgtgc tgttaaacct gtataaaggc 1140 tgtaaccctt ctgtaaaaaa aaaaaaaaaaaaa 1173 20 1134 DNA Triticum aestivum 20 ggcacgaggc ggcgcctgtcgtgcctgcag cagcagcgga gccagcagct gaatctgagc 60 cagatccaaa ccggcggcttccctcaagag ccatcccccc gcgcggcgca ctcggcgccg 120 gtcactccgc cgtcgtctggctggggaggc ctctggacgc aacaagccgt ccagagccag 180 ccccatggcc agctcatggtccaggtcccg aatccgctgt cgacgaagtc caattcctca 240 aggcagaagc agcaaaaaccctcgccggac gcagcagcga ggccgccctc cggcggcgcc 300 gccacgccgc agcgcccggggcaggcggcg gcttccgaca agcagcggca gcagggtgcg 360 aggacgccgg cggcggcgccggcggcagga gacaagaacc cgcggttcct gctgcagaag 420 gtgctcaagc agagcgacgtcggaaccctc ggccgcatcg tgctccccaa agaagcggag 480 actcacctgc cggagctcaagacgggggac ggcatctcga tccccattga ggacatcggc 540 acatctcaga ttttggcccaacaacaagag cagaatgtat cttctagaga acactggtga 600 ctttgttcgg tcgaatgagctgcaggaggg tgatttcatc gtgctttact ctgatgtcaa 660 gtcgggcaaa tatctgatacgcggcgtgaa ggtgagagcg caacaggatc tagccaagca 720 caagaatgcc agtccagagaaaggcggggc gtccgacgtg aaggcgggcg gagaagacgg 780 cggctgcaag gagaagcccccccacggcgt ccggcgatct cgccaggagg ccgcctccat 840 gaaccagatg gcggtgagcatctgaaatga gcaggctcgc cgtccgatcc accattgaag 900 actcagttag ctagctcaagtatacccgtt gatgatgatc aaatcgatct ctcgttctat 960 gatccgtgct tccgtgtactgctgtagccc tagttaggga tgatgatact aaagtagcta 1020 tcggtcagat gtgacgctgaagaatgcatg gtccgtgctg ttaaacctgt ataaaggctg 1080 taacccttct gtacatgcatgaacataccc ttaaaaaaaa aaaaaaaaaa aaaa 1134 21 1186 DNA Triticum aestivum21 cgcagcggat ggaaccggcg gcgaccaggg aggcccggaa gaagaggatg gcgaggcagc 60ggcgcctgtc gtgcctgcag cagcagcgga gccagcagct gaatctgagc cagatccaaa 120gcggcggctt ccctcaagaa ccatcccccc gcgcggcgca ctcggcgccg gtcacgccgc 180cctcttccgg ctggggaggc ctctggtcgc agcatgccgt ccagggccag ccccatggcc 240agctcatggt ccaggttccg aatccgctgt cgacgaagtc caattcctcg aggcagaagc 300agcaaaaacc ctcgccggat gcagcagcga ggccgccctc cggcggcgcc gccacgcagc 360agcgcccggg gcaggcggcg gcttccgaca agcagcggca gcagggtgcg aggacgccgg 420cggcggcgcc ggcggcagga gacaagaacc tgcggttcct gctgcagaag gtgctcaagc 480agagcgacgt cggaaccctc ggccgcatcg tgctccccaa agaagcggag actcacctgc 540cggagctcaa gacgggggac ggcatctcga tccccattga ggacatcggc acatctcagg 600tgtggagcat gcggtaccga ttttggccca acaacaagag cagaatgtat cttctggaga 660acactggaga ctttgttcgg tcgaatgagc tgcaggaggg tgatttcatc gtgctttact 720ctgatgtcaa gtcgggcaaa tatctgatac gcggcgtgaa ggtaagagcg caacaggatc 780tagccaagca caagaatggc agtccagaga aaggtggggc gtccgacgcg aaggcgggcg 840cagaagacgg tggttgcaaa gagaagtctc cgcacggtgt ccggcgatct cgccaggagg 900ccgcctccat gaaccagatg gccgtgagca tctgaaatga gcaggctcgc gcggtccgat 960cccccattga agactactta gctagctcaa gtatacctgt tgatgatgat caaatcgatc 1020tcccgttcta tgatccgtgc ttccgtgtac tgctgtagcc ctagttaggg atggtgatac 1080taaagtagct atcggtcaga tgtgacgctg aagaatgcat ggtccgtgct gttaaacctg 1140tataaaggct gtaacccttc tgttaaaaaa aaaaaaaaaa aaaaaa 1186 22 1916 DNATriticum aestivum 22 ggcacgagcc accatcgagg ccgcggccgc gcgcctcggtggggggcgcc agggcaccat 60 gcagctgctc aagctcatcc tcacctgggt gcagaaccaccacctgcaga agaagcgccc 120 ccgcgtcggc gccatggatc aggaggcgcc gccggcaggaggccagctcc ccagccccgg 180 cgcaaacccc ggctacgaat tccccgcgga gacgggtgccgccgctaaca catcttggat 240 gccctaccag gccttctcgc caactggatc ctacggcggcgaggcgatct acccgttcca 300 gcagggctgc agcacgagca gcgtggccgt gagcagccagccgttctccc cgccggcggc 360 gcccgacatg cacgccgggg cctggccgct tcagtacgcggcgttcgtcc cagctggggc 420 cacatccgca ggcactcaaa catacccgat gccgccgccgggggccgtgc cgcagccgtt 480 cgcggccccc ggattcgccg ggcagttccc gcagcggatggaaccggcgg cgaccaggga 540 ggcccggaag aagaggatgg cgaggcagcg gcgcctgtcgtgcctgcagc agcagcggag 600 ccagcagctg aatctgagcc agatccaaag cggcggcttccctcaagaac catccccccg 660 cgcggcgcac tcggcgccgg tcacgccgcc ctcttccggctggggaggcc tctggtcgca 720 gcatgccgtc cagggccagc cccatggcca gctcatggtccaggttccga atccgctgtc 780 gacgaagtcc aattcctcga ggcagaagca gcaaaaaccctcgccggatg cagcagcgag 840 gccgccctcc ggcggcgccg ccacgcagca gcgcccggggcaggcggcgg cttccgacaa 900 gcagcggcag caggtgcatg catgcacgaa cacctcttgccatccatcca tcgatcgcca 960 tcccgcatag aatcacaagc cattgctccc caaataagtgtgcgtacatc gtaagagacg 1020 cacatcgctg tccagcgata ggatatcccc gcatcgccatcccgcataga atcacaagcc 1080 attgctcccc tgcacggtga attgcgtttc tcaacgaggttccgtgcatg cgcgcagggt 1140 gcgaggacgc cggcggcggc gccggcggca ggagacaagaacctgcggtt cctgctgcag 1200 aaggtgctca agcagagcga cgtcggaacc ctcggccgcatcgtgctccc caaaaaggaa 1260 gcggagactc acctgccgga gctcaagacg ggggacggcatctcgatccc cattgaggac 1320 atcggcacat ctcaggtgtg gagcatgcgg taccgattttggcccaacaa caagagcaga 1380 atgtatcttc tggagaacac tggagacttt gttcggtcgaatgagctgca ggagggtgat 1440 ttcatcgtgc tttactctga tgtcaagtcg ggcaaatatctgatacgcgg cgtgaaggta 1500 agagcgcaac aggatctagc caagcacaag aatggcagtccagagaaagg tggggcgtcc 1560 gacgcgaagg cgggcgcaga agacggtggt tgcaaagagaagtctccgca cggtgtccgg 1620 cgatctcgcc aggaggccgc ctccatgaac cagatggccgtgagcatctg aaatgagcag 1680 gctcgcgcgg tccgatcccc cattgaagac tacttagctagctcaagtat acctgttgat 1740 gatgatcaaa tcgatctccc gttctatgat ccgtgcttccgtgtactgct gtagccctag 1800 ttagggatgg tgatactaaa gtagctatcg gtcagatgtgacgctgaaga atgcatggtc 1860 cgtgctgtta aacctgtata aaggctgtaa cccttctgtaaaaaaaaaaa aaaaaa 1916 23 414 DNA Triticum aestivum 23 ggtgcgaggacgccggcggc ggcgccggcg gcaggagaca agaacccgcg gttcctgctg 60 cagaaggtgctcaagcagag cgacgtcgga accctcggcc gcatcgtgct ccccaaaaag 120 gaagcggagactcacctgcc ggagctcaag acgggggacg gcatctcgat ccccattgag 180 gacatcggcacatctcagat tttggcccaa caacaagagc agaatgtatc ttctagagaa 240 cactggtgactttgttcggt cgaatgagct gcaggagggt gatttcatcg tgctttactc 300 tgatgtcaagtcgggcaaat atctgatacg cggcgtgaag gtgagagcgc aacaggatct 360 agccaagcacaagaatgcca gtccagagaa aggcggggcg tccgacgtga aggc 414 24 411 DNA Triticumaestivum 24 ggtgcgagga cgccggcggc ggcgccggcg gcaggagaca agaacccgcggttcctgctg 60 cagaaggtgc tcaagcagag cgacgtcgga accctcggcc gcatcgtgctccccaaagaa 120 gcggagactc acctgccgga gctcaagacg ggggacggca tctcgatccccattgaggac 180 atcggcacat ctcagatttt ggcccaacaa caagagcaga atgtatcttctagagaacac 240 tggtgacttt gttcggtcga atgagctgca ggagggtgat ttcatcgtgctttactctga 300 tgtcaagtcg ggcaaatatc tgatacgcgg cgtgaaggtg agagcgcaacaggatctagc 360 caagcacaag aatgccagtc cagagaaagg cggggcgtcc gacgtgaagg c411 25 434 DNA Triticum aestivum 25 ggtgcgagga cgccggcggc ggcgccggcggcaggagaca agaacctgcg gttcctgctg 60 cagaaggtgc tcaagcagag cgacgtcggaaccctcggcc gcatcgtgct ccccaaaaag 120 gaagcggaga ctcacctgcc ggagctcaagacgggggacg gcatctcgat ccccattgag 180 gacatcggca catctcaggt gtggagcatgcggtaccgat tttggcccaa caacaagagc 240 agaatgtatc ttctggagaa cactggagactttgttcggt cgaatgagct gcaggagggt 300 gatttcatcg tgctttactc tgatgtcaagtcgggcaaat atctgatacg cggcgtgaag 360 gtaagagcgc aacaggatct agccaagcacaagaatggca gtccagagaa aggtggggcg 420 tccgacgcga aggc 434 26 431 DNATriticum aestivum 26 ggtgcgagga cgccggcggc ggcgccggcg gcaggagacaagaacctgcg gttcctgctg 60 cagaaggtgc tcaagcagag cgacgtcgga accctcggccgcatcgtgct ccccaaagaa 120 gcggagactc acctgccgga gctcaagacg ggggacggcatctcgatccc cattgaggac 180 atcggcacat ctcaggtgtg gagcatgcgg taccgattttggcccaacaa caagagcaga 240 atgtatcttc tggagaacac tggagacttt gttcggtcgaatgagctgca ggagggtgat 300 ttcatcgtgc tttactctga tgtcaagtcg ggcaaatatctgatacgcgg cgtgaaggta 360 agagcgcaac aggatctagc caagcacaag aatggcagtccagagaaagg tggggcgtcc 420 gacgcgaagg c 431 27 500 DNA Triticum aestivum27 tcacaagcca ttgctcccct gcacggtgaa ttgcgtttct caacgaggtt ccgtgcatgc 60gcgcagggtg cgaggacgcc ggcggcggcg ccggcggcag gagacaagaa cctgcggttc 120ctgctgcaga aggtgctcaa gcagagcgac gtcggaaccc tcggccgcat cgtgctcccc 180aaaaaggaag cggagactca cctgccggag ctcaagacgg gggacggcat ctcgatcccc 240attgaggaca tcggcacatc tcaggtgtgg agcatgcggt accgattttg gcccaacaac 300aagagcagaa tgtatcttct ggagaacact ggagactttg ttcggtcgaa tgagctgcag 360gagggtgatt tcatcgtgct ttactctgat gtcaagtcgg gcaaatatct gatacgcggc 420gtgaaggtaa gagcgcaaca ggatctagcc aagcacaaga atggcagtcc agagaaaggt 480ggggcgtccg acgcgaaggc 500 28 11 PRT Triticum aestivum 28 Thr Ser Gln ValTrp Ser Met Arg Tyr Arg Phe 1 5 10 29 20 DNA Artificial SequenceDescription of Artificial Sequence Oligonucleotide 29 caactcatggtcccgaatcc 20 30 19 DNA Artificial Sequence Description of ArtificialSequence Oligonucleotide 30 gcttgttaga cgaattgac 19 31 26 DNA ArtificialSequence Description of Artificial Sequence Primer 31 aatatctgatacgcggcgtg aaggtg 26 32 25 DNA Artificial Sequence Description ofArtificial Sequence Primer 32 aggatctagc caagcacaag aatgg 25 33 25 DNAArtificial Sequence Description of Artificial Sequence Primer 33gcccatatga actcgatcga ttgac 25 34 26 DNA Artificial Sequence Descriptionof Artificial Sequence Primer 34 gttgtccata tgaactcgat cgattc 26

What is claimed is:
 1. An isolated nucleic acid molecule encoding Avenafatua viviparous 1 (afVP1) comprising a nucleotide sequence identical toSEQ ID NO: 1 or degeneratively equivalent thereto.
 2. A nucleic acidwhich is the complement of the nucleic acid of claim
 1. 3. A recombinantvector comprising the nucleic acid of claim
 1. 4. A host cell comprisingthe vector as claimed in claimed
 3. 5. A method for transforming a plantcell, comprising the step of introducing the vector as claimed in claim3 into a cell, and causing or allowing recombination between the vectorand the plant cell genome to introduce the nucleic acid into the genome.6. A method for producing the transgenic plant comprising a method asclaimed in claim 5 and further comprising the step of regenerating aplant from the transformed cell.
 7. A method of producing a polypeptidecomprising the step of causing or allowing the expression of the nucleicacid of claim 1 in a suitable host.
 8. A method for increasing thedormancy of a seed or grain, the method comprising the step of causingfor allowing expression of the nucleic acid according to claim 1 withinthe seed or grain.
 9. A wheat plant comprising the cell of claim 4.